Methods and compositions for preventing and treating a disease related to glycan dysregulation

ABSTRACT

Contemplated compositions and methods are directed to prevent and/or treat various autoimmune diseases that are typically associated with glycan dysregulation, and especially autoimmune demyelinating diseases. Further especially contemplated aspects include animal models and systems for screening compounds to treat and/or prevent such diseases.

This application is a divisional of U.S. patent application Ser. No.11/795,183, filed Oct. 3, 2008, which is a US national phase ofInternational Patent Application No. PCT/US2006/001337, filed Jan. 13,2006, which claims the benefit of the priorities of U.S. ProvisionalPatent Application No. 60/663,380, filed Mar. 17, 2005, and U.S.Provisional Patent Application No. 60/643,962, filed Jan. 14, 2005. Eachof these applications is hereby incorporated by reference.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of diagnosis,prevention, and treatment of diseases associated with glycandysregulation, and especially as they relate to autoimmune diseases.Further aspects are directed to compositions and methods of animalmodels and systems for screening compounds to diagnose, treat and/orprevent such diseases.

BACKGROUND OF THE INVENTION

Multiple Sclerosis (MS) is a T-cell mediated autoimmune demyelinatingdisease of the central nervous system (CNS) of unknown etiology (1-3),and often presents in several clinically distinct forms. For example,relapsing remitting multiple sclerosis (MS) is characterized by T-cellinduced autoimmune destruction of the myelin sheath and producesrelapsing and remitting attacks of neurological dysfunction (RRMS)(1-3), which is typically followed by a secondary progressiveneurodegenerative phase (SPMS), distinguished by axonal damage andneuronal loss (2). Primary progressive MS (PPMS) is similar to SPMS butlacks the initial relapsing-remitting phase.

With adult onset and partially familial relationships, causality isthought to result from complex interactions between environmental andgenetic factors (1,6,7). Whole genome screens have identified a numberof candidate loci associated with MS (8) and the animal model EAE (9,10), which are typically MHC-related genes. However, non-MHC genes withstrong association to MS have yet to be identified as such genes maycorrelate to the suspected environmental component of the MS etiology.

Golgi β1-6 N-acetylglucosaminyltransferase V (Mgat5) is a potentnegative regulator of TCR signaling, T-cell proliferation, TH1differentiation and autoimmunity (e.g., involved in EAE, a murine modelof MS) (4,5). Mgat5-modified N-glycans are extended withpoly-N-acetyllactosamine sequences, which are preferred ligands forgalectins. Mgat5 N-glycans on T cell receptor (TCR) hind multi-valentgalectins, thus restricting TCR recruitment into the immune synapse (4).It should be noted that myelin-specific transgenic mice developspontaneous CNS autoimmune demyelinating disease (11-14), butspontaneous disease secondary to physiologically-relevant genedysfunction has not been reported.

Diagnosis of MS is often based on several individual potential markers,and most commonly focus on identification of specific nucleic acids asdescribed in U.S. Pat. No. 6,933,119 to Leppert et al. or U.S. Pat. No.6,001,978 to Perron. Other known diagnostic assays involve detection ofspecific polypeptides as described by Kline in U.S. Pat. No. 5,883,227,or on T-cell subpopulation measurement as taught in U.S. Pat. No.4,677,061. Still further known diagnostic tests are based on tests ofhuman motor function as described in U.S. Pat. No. 6,702,756.

Similarly, numerous chemically distinct treatment modalities for MS havebeen proposed. Among many other compounds, modified adenine derivativeswere employed as described in U.S. Pat. No. 5,506,214, whileheterocyclic phospholipids were proposed as therapeutic agents in U.S.Pat. No. 5,064,816. In yet other examples of pharmaceutical activecompounds for treatment of MS, chloroquine was presented in U.S. Pat.No. 5,624,938, and aloe vera products were described as therapeuticagents in U.S. Pat. No. 5,780,453. Still further known compositions fortreatment of MS include peptide analogues as taught in U.S. Pat. No.5,948,764, estriol as described in U.S. Pat. No. 6,936,599, and varioustetracycline derivatives as taught in U.S. Pat. No. 6,613,756.

As will be readily apparent to the person of ordinary skill in the art,such vast disparity in proposed active ingredients for treatment of MSand disparate markers strongly suggest a multi-factorial etiology, apotentially complex underlying metabolic system, and/or a general lackin the interplay of environmental factors and underlying geneticdisposition.

Thus, while numerous compositions and methods for diagnosis, prevention,and treatment of MS and other autoimmune diseases are known in the art,all or almost all of them, suffer from one or more disadvantages.Therefore, there is still a need for improved pharmaceutical agents fortreatment and chemoprevention of MS and other autoimmune diseases.

SUMMARY OF THE INVENTION

Applicants using forward and reverse genetic methods have demonstratedthat a biochemical deficiency of the Golgi processing pathway leading toβ1,6GlcNAc branched tetrantennary N-glycan predisposes to autoimmunediseases. A nonhuman animal model of autoimmune demyelinating diseases,more particularly multiple sclerosis (MS), was developed. DefectiveN-glycan processing induces spontaneous demyelinating disease in theanimal model and metabolically supplementing the hexosamine pathwayrescues this phenotype and inhibits disease by increasing supply ofUDP-GlcNAc to MGAT5.

The invention also revealed that the PL/J mouse strain background isnaturally hypomorphic for production of β1,6GlcNAc branchedtetrantennary N-glycan, and is an aspect of the disease model that isadditive with mutation of Mgat5.

In an aspect, the invention provides a nonhuman animal characterized bylacking one or both Mgat5 gene alleles at least in its somatic cells anddisplaying pathology of an autoimmune demyelinating disease, inparticular a CNS demyelinating disease, more particularly MS. In aspectsof the invention the incidence, severity, and/or mortality of disease inthe nonhuman animal are characterized by an inverse correlation withMgat5 N-glycan products.

In another aspect, the invention relates to methods for generatingnonhuman animals of the invention. In addition, the invention relates tomethods of using a nonhuman or transgenic animal of the invention as amodel animal of an autoimmune demyelinating disease, in particularmultiple sclerosis, comprising measuring the extent of presentation ofcharacteristics similar to an autoimmune demyelinating disease, inparticular multiple sclerosis, more particularly PPMS and SPMS.

The invention further relates to a transgenic nonhuman animal assaysystem which provides a model system for testing a compound that reducesor inhibits pathology associated with a condition or disease describedherein. Therefore, in an aspect the present invention provides methodsof screening a test compound comprising exposing a nonhuman animal ofthe invention to the test compound; and determining a response of theanimal to the test compound. In yet another aspect, the presentinvention provides a method of conducting a drug discovery businessusing the methods for screening compounds as described herein.

Compounds identified using methods of the invention may be useful in thetreatment and prophylaxis of diseases discussed herein. The compoundsmay also be incorporated in pharmaceutical compositions.

Applicants have also demonstrated that an autoimmune disease such as MSis associated with inherent genetic deficiencies in the N-glycan pathwaythat reduce β1, 6 GlcNAc branching and promote autoimmunity conditionalto metabolite flux through the hexosamine pathway. N-glycan andhexosamine pathway genes are over represented at putative MS loci.Glycomic analysis led to the identification of rare MS associated singlenucleotide polymorphisms (SNPs) within the N-glycan pathway.

In one aspect of the inventive subject matter, Applicants found thatMgat5 glycan expression in cells obtained from MS patients wassignificantly lowered as compared to normal control cells obtained fromhealthy patients, and that such reduction in Mgat5 glycans is correlatedin a gradual manner with T-cell receptor sensitivity and susceptibilityto spontaneous and induced demyelinating disease. Contrary totraditional approaches, Applicants then investigated all genes known toinfluence and/or regulate N-glycan biosynthesis leading to β1,6GlcNAcbranched tetrantennary N-glycan, that is Mgat5-modified N-glycans, anddetermined if such genes indeed correlate with linkage mapping for MS.In a further step, they probed the structural glycan products ofassociated pathways to identify abnormal accumulation and/or depletionof the components (and their substrates and products). Based on suchanalysis, genes were sequenced to detect defects that would beassociated with the disease, and the findings were then correlated withfunctional assays to identify mutations and/or polymorphisms that atleast in part contribute to the disease.

Broadly stated, the invention contemplates a method comprising two,three, or more, preferably all, of the following steps:

-   -   a) Identify one or more defective biochemical pathway using        model systems and validate with human tissue.    -   b) Bioinformaticly identify all potential human genes that may        regulate the pathway(s) and determine whether they map to loci        associated with the disease identified by traditional gene        mapping techniques.    -   c) Obtain relevant patient tissue and use mass spectroscopy to        identify abnormal accumulation of pathway intermediates.    -   d) Sequence all genes that regulate identified defects in the        pathway(s) starting with genes that map disease.    -   e) Validate identified mutations/polymorphisms with functional        assays and determine frequency in control and patient        populations.

More specifically, it was found that Mgat5 glycans are reduced onresting T cells and display reduced up-regulation following TCRstimulation. Thus, a dysregulation of Mgat5 in which Mgat5 quantityand/or activity is reduced promotes an undesirable reduction in GlcNAcbranched N-glycans. Natural genetic variations that reduce expression ofMgat5-modified glycan increase sensitivity to autoimmune disease. Usingthe broad method as outlined above it was found that a number of genesin the N-glycan pathway required for Mgat5 glycan expressiondisproportionately mapped to known MS associated loci, and that limitedenzymatic inhibition of these genes phenocopy MS T cells. SubsequentMALDI-TOF mass spectroscopy of glycans from patients with MS showed thatpatients with MS frequently have blocks at various steps in the N-glycanprocessing pathway. Based on these and other facts, numerous singlenucleotide polymorphisms were discovered in the relevant genes that areblocked in the N-glycan pathway as defined by MALDI-TOF. Taken together,these data identify biochemical and genetic defects in the N-glycanprocessing pathway as significant susceptibility factors in MS andsuggest that these genes are likely to be defective in other autoimmunediseases.

It is therefore contemplated that numerous diseases, and especiallyautoimmune diseases (e.g., MS and rheumatoid arthritis) may be diagnosed(in some cases even before first manifestation of signs and symptoms) byanalyzing one or more component of implicated pathways with a gene knownto be associated with a disease.

For example, genetic testing may be employed to identify mutations thatfunctionally affect (in terms of control or level of expression as wellas in terms of coding for a dysfunctional mutant protein) at least one,and more typically most or all of the components in a pathway known toinclude a gene that is relevant to a disease. Such testing may be doneusing solid-phase based testing (e.g., gene chip) for SNPs, deletions,and/or other mutations. Alternatively, genetic testing may also includertPCR and/or quantitative PCR to determine the level of expression ofthe genes that are part of the pathway.

Additionally, or alternatively, testing may also include all factorsthat are known to regulate directly or indirectly expression of a genein a suspect pathway. For example, vitamin D is known to affectexpression of one or more genes in the Mgat5 pathway. Similarly,substrate concentrations of enzymes may be determined in a patientsample, where that enzyme is known to catalyze a rate-limiting step inthe pathway.

While testing may be performed on the protein, carbohydrate, or cellularlevel, all analytical methods well known to the art may be employed. Forexample, expression levels may be quantitated using antibodies or theirfragments (e.g., via ELISA, western blot, FACS, etc.), by determinationof enzymatic kinetics of the polypeptides (e.g., K_(M), kcat, etc.) orby mass spectroscopy analysis of the proteins or glycans in biologicalsamples. Similarly, it is also contemplated that a patient sample may beanalyzed for the presence or quantity of a substrate, product, and/orcofactor that is required by an enzyme that is part of the pathway underinvestigation. For example, the quantity of Mgat5 glycan product may bedetermined in patients that are diagnosed for MS.

Based on the diagnostic outcome, suitable treatment options may then bedevised. For example, where diagnostic testing has revealed a lowconcentration of a substrate for a rate-limiting step in an enzymaticcascade, supplementation (dietary or otherwise) may be useful intreatment of a disease. Similarly, where diagnostic testing has revealeda low concentration of a compound that induces expression of a keyenzyme (e.g., Vitamin D in Mgat5), supplementation (dietary orotherwise) may be useful in treatment of a disease.

Alternatively, genetic defects may also be overcome by somatic genetherapy in which a delivery vehicle (e.g., viral, or liposomal) providesone or more genes to counterbalance or correct the underlying geneticdefect. Of course, where possible, the underlying genetic defect mayalso be treated by targeting the element that corresponds to the defect.For example, where a lack of the product of Mgat5 triggers T-cellactivation, products and/or metabolites that raise Mgat5 enzyme or Mgat5modified glycans may be employed as therapeutic agents.

Specific embodiments and applications of compositions and methodsrelated to glycan dysregulation and associated conditions are disclosedherein.

In an aspect, the invention provides methods and compositions fortreating or preventing a disease discussed herein, in particular anautoimmune disease, more particularly and rheumatoid arthritis or anautoimmune demyelinating disease, most particularly multiple sclerosis,in a subject comprising increasing in the subject expression or amountof N-glycans, in particular Mgat5 modified glycans and/orpolylactosamine modified glycans. The expression or amount of N-glycans,in particular Mgat5 modified glycans or polylactosamine modifiedglycans, can be increased by administering N-glycans (e.g., Mgat5modified glycans or polylactosamine modified glycans) to the subject oran agonist of a component of the N-glycan or hexosamine pathways (e.g.,an agonist of an enzyme of the N-glycan pathway, especially Mgat5), orincreasing expression or synthesis of a component of the N-glycan orhexosamine pathways (e.g., an enzyme of the N-glycan pathway, especiallyMgat5), an acceptor for an enzyme of the N-glycan pathway (e.g., anacceptor for Mgat5), or a donor for an enzyme of the N-glycan pathway(e.g., a donor for Mgat5) or metabolites thereof.

In another aspect, the invention provides a method of treating anautoimmune disease in a subject comprising modulating one or more ofN-glycan processing, an N-glycan pathway, a hexosamine pathway, and/orN-glycans (e.g., expression or levels), in particular Mgat5 glycans orpolylactosamine modified glycans. In an aspect, N-glycan processing, anN-glycan pathway, a hexosamine pathway, and/or N-glycans (e.g.,expression or levels), in particular Mgat5 glycans or polylactosaminemodified glycans, are modulated by modulating one or more of glucosidaseI (GCS1), glucosidase, alpha, neutral AB (GANAB), glucosidase II,mannosidase I (MI), Mannosidase, alpha, class 1A, member 1 (MAN1A1),mannosidase II (MII/MIIx), MGAT1, and MGAT5. In a particular embodiment,N-glycan processing, the N-glycan pathway, the hexosamine pathway,and/or N-glycans (in particular Mgat5 modified glycans), are modulatedby administering a substance that raises N-glycan levels or up-regulatesMGAT5 expression. In an embodiment, the method comprises increasingMgat5 modified glycan levels by administering an agonist of Mgat5, asugar donor for Mgat5, a metabolite of pathways for synthesis of thesugar donor or precursors thereof (e.g., a hexosamine pathwaymetabolite), or regulators of agonists of a sugar donor or pathway forsynthesis of a sugar donor.

In another aspect, the invention provides a composition, in particular apharmaceutical composition, comprising (a) an agonist of one or more ofthe following enzymes: glucosidase I (GCS1), glucosidase, alpha, neutralAB (GANAB), glucosidase II, mannosidase I (MI), Mannosidase, alpha,class 1A, member 1 (MAN1A1), mannosidase II (MII/MIIx), Mgat1, Mgat2,and Mgat5; or (b) a sugar donor for one of the enzymes in (a) or ametabolite of pathways for synthesis of the sugar donor or precursorsthereof, or regulators of agonists of a sugar donor or pathway forsynthesis of a sugar donor.

In an embodiment, the invention provides a composition, in particular apharmaceutical composition, comprising an agonist of Mgat5, a sugardonor for Mgat5, a metabolite of pathways for synthesis of the sugardonor or precursors thereof, or regulators of agonists of a sugar donoror pathway for synthesis of a sugar donor.

A pharmaceutical pack or kit is provided comprising one or morecontainers filled with one or more of the ingredients of a compositionof the invention to provide a therapeutic effect. Associated with suchcontainer(s) can be various written materials such as labels,instructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, dietary supplements, or biological products, whichnotice reflects approval by the agency of manufacture, use, or sale forhuman administration.

The invention provides a method of treatment or prophylaxis of a diseasedisclosed herein (e.g., an autoimmune disease) based on the presence ofa polymorphism in a gene of the N-glycan pathway or hexosamine pathway.

The invention also provides a method for treating a disease disclosedherein (e.g., an autoimmune disease) comprising obtaining a sample ofbiological material containing at least one polynucleotide from thesubject; analyzing the polynucleotide to detect the presence of at leastone polymorphism in a gene of the N-glycan pathway or hexosamine pathwayassociated with the disease; and treating the subject in such a way asto counteract the effect of any such polymorphism detected.

In an aspect of the invention, a method is provided for the prophylacticprevention of a subject with a genetic predisposition to a diseasedisclosed herein, (e.g., an autoimmune disease) comprising obtaining asample of biological material containing at least one polynucleotidefrom the subject; analyzing the polynucleotide to detect the presence ofat least one polymorphism in a gene of the N-glycan pathway orhexosamine pathway associated with the disease; and treating thesubject.

The invention provides methods, reagents and kits for detecting anindividual's increased or decreased risk for a disease disclosed herein,in particular autoimmune and related diseases. Therefore, the inventioncontemplates methods of, and products for, diagnosing and monitoring ofa disease disclosed herein (e.g, an autoimmune disease, in particular anautoimmune demyelinating disease, more particularly multiple sclerosis)in a sample from a subject, comprising assaying for an alteration orchange in an N-glycan (e.g., Mgat5 modified glycans or polylactosaminemodified glycans), and/or in polypeptides or genes of a N-glycan pathwayor hexosamine pathway (e.g, Mgat5), in a sample from the subjectcompared to a standard.

The invention provides a method of analyzing a polynucleotide from anindividual to determine which nucleotides are present at polymorphicsites within a gene of the N-glycan pathway or hexosamine pathway. Theanalysis can be performed on a plurality of individuals who are testedfor the presence of the disease phenotype. The presence or absence of adisease phenotype or propensity for developing a disease state can thenbe correlated with a base or set of bases present at the polymorphicsites in the individual tested. Alternatively, this determination stepis performed in such a way as to determine the identity of thepolymorphisms.

The invention relates to methods for using polymorphisms associated witha gene of the glycan pathway or hexosamine pathway to diagnose a diseasedisclosed herein (e.g, an autoimmune disease).

In an aspect the invention provides a method for diagnosing or aiding inthe diagnosis of a disease disclosed herein (e.g, an autoimmune disease)in a subject comprising the steps of determining in the subject thegenetic profile of genes of an N-glycan pathway or a hexosamine pathway(in particular genes co-localized in chromosomal regions associated withthe disease), thereby diagnosing or aiding in the diagnosis of thedisease.

In an aspect the invention provides a method for diagnosing a geneticsusceptibility for a disease disclosed herein (e.g, an autoimmunedisease) in a subject comprising obtaining a biological samplecontaining polynucleotides from the subject; and analyzing thepolynucleotides to detect the presence or absence of a polymorphism in agene of a N-glycan pathway or hexosamine pathway of the subject whereina polymorphism is associated with a genetic predisposition for thedisease.

The invention provides a method for screening patients comprisingobtaining sequence information for one or more genes of the N-glycanpathway or hexosamine pathway from the patient and determining theidentity of one or more polymorphisms in the gene(s) that is indicativeof a disease disclosed herein (e.g, an autoimmune disease). The patientmay be at risk of developing the disease or have the disease.

The invention also provides a method for determining the efficacy of atreatment for a particular patient with a disease disclosed herein(e.g., an autoimmune disease) based on genotype comprising (a)determining the genotype for one or more polymorphism sites in a gene ofthe N-glycan pathway or hexosamine pathway for a group of patientsreceiving a treatment; (b) sorting patients into subgroups based ontheir genotype; (c) identifying correlations between the subgroups andthe efficacy of the treatment in the patients, (d) determining thegenotype for the same polymorphism sites in the gene(s) of theparticular patient and determining the efficacy of the treatment for theparticular patient based on a comparison of the genotype with thecorrelations identified in (c).

The invention further provides a method for classifying a subject who isor is not at risk for developing a disease disclosed herein (e.g., anautoimmune disease) as a candidate for a particular course of therapy ora particular diagnostic evaluation. The invention still further providesa method for selecting a clinical course of therapy or a diagnosticevaluation to treat a subject who is or is not at risk for developing adisease disclosed herein (e.g, an autoimmune disease).

The invention also relates to a kit for carrying out a method of theinvention.

These and other aspects, features, and advantages of the presentinvention should be apparent to those skilled in the art from thefollowing drawings and detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1. Dystonic posturing and CNS/PNS Demyelinating Pathology in PL/Jmice. A) Clinically affected mice were observed to have dystonicposturing of the tail, hind limbs and/or axial skeleton. B-E) Paraffinembedded sections from brainstem (B), spinal cord (C,E) and spinal roots(D) from clinically affected PL/J mice were stained with Haematoxylin &Eosin (E) or Luxol Fast Blue (B-D). Arrows point to large naked axons.F) Frequency of spinal root (PNS) and CNS pathology in Mgat5^(+/+)(n=9), Mgat5^(+/−) (n=10) and Mgat5^(−/−) (n=17) PL/J mice (p=0.048, chisquare for CNS).

FIG. 2. Mgat5^(−/−) T-cells have intermediate Mgat5 glycan expressionand TCR sensitivity A) Mgat5 modified glycans and binding of L-PHA, LEAand galectin B,C) FACS analysis with L-PHA-FITC of resting splenocytes(A) and 3 day anti-CD3 stimulated (B) CD3+ T cells from PL/J mice withthe indicated genotypes. Data shown gated on CD4⁺ population. Error barsin C) are standard error of triplicate staining. D) Purified CD3⁺ PL/JT-cells were labeled with CFSE, stimulated for 72 hrs as indicated andanalyzed by FACS. Plots shown are gated on CD4⁺ cells. E) Purified CD3⁺T-cells were incubated at 37° with anti-CD3 antibody coated beads forvarious times, lysed and western blotted for the indicatedphosphorylated proteins. Reduced phosphorylation of lck at 3 minutesrelative to rest is likely secondary to clustering of the lckphosphatase CD45 at the immune synapse during this time.

FIG. 3. Differential expression of Mgat5 glycans in multiple inbredstrains of mice. A-C) Mgat5^(+/+) (A-C), Mgat5^(+/−) (A) and Mgat5^(−/−)(A) resting splenocytes (A,B) or CD3+ T-cells (C) from the indicatedinbred mouse strains were stained with L-PHA-FITC and anti-CD4 (A,B),anti-CD8 (B) or anti-13220 (13) antibodies and analyzed by FACS (A,B) orused to isolate mRNA for cDNA synthesis and analysis by quantitativereal time-PCR (C) (5). Shown in B and C is relative expressionnormalized to wild type 129/Sv cells. Data in C are averaged from twomice for each genotype repeated once. Error bars refer to standard error(A-C). D) Purified CD3⁺ T-cells from Mgat5^(+/−) and Mgat5^(−/−) 129/Svand PL/J mice were stimulated with anti-CD3 antibody coated beads forthe specified times and western blotted for the indicated proteins.

FIG. 4. Metabolic regulation of Mgat5 glycan expression, T cell functionand EAE by the hexosamine pathway. A) Hexosamine pathway andbiosynthesis of UDP-GlcNAc, the sugar nucleotide donor for Mgat5. B-D)The indicated monosaccharides and metabolites were cultured with JurkatT-cells for 3 days (D), stained with L-PHA-FITC or LEA-FITC, a lectinspecific for poly-N-acetyllactosamine, and analyzed by FACS (B,D) orlysed and analyzed by MS/MS mass spectroscopy for sugar-nucleotideexpression (C). Lines with symbols ▪, ▴, ▾ refer to altered glucoseconcentration in the culture media as indicated; all others were grownin 10 mM glucose. Error bars in D are standard error of triplicatestaining. E, F) PL/J wild type CD3⁺ T cells left unlabelled (E) orlabeled with CFSE (F) were stimulated with anti-CD3 antibody,swainsonine and/or GlcNAc as indicated for 3 days and analyzed by FACSfor L-PHA (E) and CFSE staining (F). Arrow defines undivided cellpopulation. Shown are gated on CD4+ cells. G-I) Splenocytes isolatedfrom PL/J wild type mice 11 days following immunization with MBP+CFAwere re-stimulated in vitro with MBP for 4 days in the presence (lightgray) or absence (dark gray) of GlcNAc (40 mM), stained with L-PHA-FITCand LEA-FITC (G), tested for IFNγ secretion in harvested supernatant (H)and injected into naïve Mgat5^(+/−) PL/J mice (n=7 for each condition)and scored for signs of EAE daily for 30 days (1). Shown in H isstandard error of duplicate values. I) P values for disease incidenceand mean clinical score was determined by Fishers exact test and theMann-Whitney nonparametric test, respectively.

FIG. 5. Demyelinating and axonal pathology and Electromyography andNerve Conduction Studies in PL/J mice. A-F) Paraffin embedded sectionswere stained with Haematoxylin & Eosin (A-C,F) or Luxol Fast Blue (D-E).A-B) Shows spinal cord demyelination, gliosis and neuronophagia. C)Shows axonal swelling in spinal cord surrounded by otherwise normalappearing white matter. D) Shows multi-focal myelin degeneration ofspinal roots. E) Shows neuronal bodies with central chromatolysis in thespinal cord. F) Shows spinal root with swollen axons. G-H) PL/J miceunderwent needle electromyography (EMG) and nerve conduction studies forassessment of F waves and H responses, a clinical physiological test forspinal root demyelination. G) Example of positive sharp waves observed.H) Frequency of delayed or absent F and H responses in mice with normaland abnormal needle EMG.

FIG. 6. Intermediate T cell activation thresholds in Mgat5^(+/−) 129/SvT cells and additive effect of Uridine plus GlcNAc. A) Purified CD3⁺129/Sv T-cells were labeled with CFSE, stimulated for 72 hrs asindicated and analyzed by FACS. Plots shown are gated on CD⁺ cells. B,C)Jurkat T cells were incubated as indicated for 3 days and stained withL-PHA-FITC and analyzed by flow cytometry. Error bars are standard errorof triplicate staining. D) Wildtype C57/B6 CD3⁺ T cells labeled withCFSE were stimulated with anti-CD3 antibody, swainsonine and/or GlcNAcas indicated for 3 days and analyzed by FACS.

FIG. 7. Regulation of Mgat5 glycan synthesis by the N-glycan andhexosamine pathways. A) Regulation of β1, 6GlcNAc-branched N-glycanbiosynthesis by the Hexosamine and N-glycan pathways. Genes shown areinvolved in the production and Golgi transport of UDP-GlcNAc as well asN-glycan biosynthesis to β1, 6GlcNAc-branched N-glycans withpoly-N-acetyllactosamine. Those in blue localize to 18 putative MS loci(Table 4, 5). UDP-GlcNAc is required by theN-acetylglucosaminyltransferases MGAT1, 2, 3, 4 & 5 and B3GNT4 & 6. β1,6 GlcNAc branching by MGAT5 promotes poly-N-acetyllactosamine productionby B3GNT4 & 6 and the galactosyltransferase B4GALT3, forming a highaffinity ligand for galectins. MGAT3 negatively regulates β1,6GlcNAc-branched N-glycan levels by producing a bisecting GlcNAc thatinhibits MII (MAN2A1), MGAT2 and MGAT5 activity (59). All enzymes aremonomeric except GII (two subunits) and OT (multiple subunits), where atleast one subunit, GCS1, GANAB and DDOST respectively, map to one of the18 MS regions. B,C) mRNA isolated from Jurkat T cells at rest andstimulated with anti-CD3 antibody for the indicated doses and times wasreverse transcribed into cDNA and analyzed by Taqman quantitativeRT-PCR. B) Relative expression of the indicated genes at rest,normalized to MAN2A1. C) Change in mRNA expression of the indicatedgenes following TCR stimulation normalized to the resting state. D)Jurkat T cells were incubated with the alkaloids castanospermine (CST),deoxymannojirimycin (DMN) or swainsonine (SW) for 3 days to inhibitvarious steps in N-glycan processing (see A) and stained with L-PHA-FITCand analyzed by FACS. Shown is the relative change in staining comparedto untreated. Error bars are S.E.M for triplicate staining.

FIG. 8. N-Glycan processing in controls and MS patients. A) N-glycanprocessing pathway demonstrating Golgi intermediates and associatedmonoisotopic mass (permethylated, Na⁺) on MALDI-TOF mass spectroscopy.Glycan species were grouped based on enzymatic function. Structures ingroup A were found as 4, 5 and 6 hexose fragments as shown. Mass species2396, 1662, 1907 and 2153 were not reliably detected in control samplesand not included in the analysis. B) MALDI-TOF mass spectroscopy profileof control 1 (C1) and MS patient 1 (PA1) with the peaks labeled with theglycans they represent. C) The relative intensity of structures A-G forcontrols and MS patients were obtained by adding the intensity ofstructures from each group and dividing by the combined intensity of themature bi-antennary (2071) and tri-antennary (2520) glycans. Coefficientof determination R² values were derived by averaging the relativeintensity of each glycan structure group from the 5 controls andcomparing this to each MS patient. The presence of the abnormal glycanprofiles in 6/7 patients and 0/5 normal controls is associated with MS(p=0.0152, Fishers exact test).

FIG. 9. Association of novel single nucleotide polymorphisms in genescontrolling obstructed N-glycan processing in MS patients. A) MSpatients with altered N-glycan processing are shown along with the genescontrolling the defective steps. Sequencing of genomic DNA derived PCRproducts for each exon identified multiple heterozygous and homozygousunknown (red) and previously identified SNPs (blue, NCBI SNP database)as indicated. Exon and introns are not to scale. Controls C1-5 are shownas number of chromosomes with each SNP as determined by DNA sequencingand/or allelic discrimination. Pop. Het. refers to predictedheterozygosity of each SNP as defined in the NCBI SNP database. MAN1A2and MAN1C1 were not targeted for sequencing as the former does not mapto the 18 MS loci and the latter is not significantly expressed in Tcells (FIG. 7B) (See Tremblay, L.O. & Herscovics, A. Characterization ofa cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involvedin N-glycan biosynthesis. J. Biol. Chem. 275, 31655-31660 (2000)).Sequences for the previously unknown SNPs are in FIG. 11. The known SNPsare GCS1: II-rs1063588, III-rs2268416; GCS1, GANAB: I-rs2957121,11-rs11231168, III-rs10897289, V-rs11231166; MAN1A1: I-rs6915947,II-rs195092, III-rs9481891, IV-rs2072890, V-rs2142887, VI-rs3756943,VII-rs18513744, VIII-rs3798602, IX-rs1042800, XI-rs1046226. MGAT1:I-rs3733751, II-rs7726357, III-rs2070924, IV-rs2070925, V-rs634501.MGAT5: III-rs3214771, IV-rs3748900, V-rs2289465, B) Table showingpresence or absence of rare SNPs in genes controlling the obstructedsteps for the 7 MS patients and 5 controls. Rare SNPs are defined aspresent in one or more individuals at the obstructed step and wereeither previously unknown or possess an allele frequency of ≦4%.

FIG. 10. Reduced Mgat5 glycan expression in MS patient T cells and withblockade of glycosidase activity. A) Resting PBMC from the 7 MS patientsand 5 controls were directly compared for Mgat5 glycan expression byL-PHA-FITC staining and flow cytometry analysis; shown is gated on CD4⁺population. B) Freshly isolated PBMC were left unstimulated orstimulated with anti-CD3 antibody or a mixture of myelin antigens for 48hrs and analyzed for L-PHA staining levels. Fold change in L-PHA MFI wascalculated by comparing the MFI of blasting cells vs unstimulated cellsas defined by side vs forward scatter in stimulated and non-stimulatedcultures. Shown is gated on CD4⁺ population. C) Jurkat T cells wereuntreated or stimulated with PMA and ionomycin in the presence orabsence of minimal concentrations of the indicated glycosidaseinhibitors for three days and stained with L-PHA-FITC. D) Mouse T cellspurified by negative selection (R&D) were left unstained or stained withCFSE and stimulated with anti-CD3 antibody in the presence or absence ofthe indicated doses of SW for 5 days. Shown is the L-PHA MFI fornon-CFSE labeled cells (top panel) and the percentage increase in thenumber of proliferating CD4⁺ T cells relative to cells stimulated at62.5 ng/ml anti-CD3 (bottom panel). Proliferating vs non-proliferatingcells were determined by defining the latter with a gate on CFSE labeledcells not stimulated with antibody. E-G) Jurkat T cells were treatedwith the indicated concentrations of SW/CST (E), DMN in the absence orpresence of GlcNAc (20 mM) and Uridine (10 mM) (E) orAnti-CD3+/−1α25-dihydroxyvitamin D3 for 3 days and analyzed by FACS forL-PHA staining.

FIG. 11. Rare SNPs in GCS1, GCS1, GANAB, MAN1A1, MGAT1 and MGAT5associated with MALDI-TOF Glycan profile. A-I) Sequences are shown forthe indicated SNPs with small, unlabeled boxes defining coding SNPs,arrow heads defining UTR and intronic SNPs and exon/intron junctions asindicated. A) GCS1 SNP 1 (Exon 1): The DNA sequence for the genomicsequence of GCS1 Exon I is shown in SEQ ID NO: 48. The correspondingamino acid sequence is shown in SEQ ID NO: 49. The DNA sequence formultiple sclerosis patient PA3 GCS1 Exon I (containing SNP I) is shownin SEQ ID NO: 50. The corresponding amino acid sequence is shown in SEQID NO: 51. B) GANAB SNP III (Exon 11): The DNA sequence for the genomicsequence of GANAB Exon 11 is shown in SEQ ID NO: 52. The correspondingamino acid sequence is shown in SEQ ID NO: 53. The DNA sequence forpatient PA3 GANAB Exon 11 (containing SNP III) is shown in SEQ ID NO:54. The corresponding amino acid sequence is unchanged and is shown inSEQ ID NO: 53. C) GANAB SNP IV (Intron 21): The DNA sequence for thegenomic sequence of GANAB Intron 21 is shown in SEQ ID NO: 55. The DNAsequence for patient PA1 GANAB Intron 21 (containing SNP IV) is shown inSEQ ID NO: 56. D) GANAB SNP V (Intron 23): The DNA sequence for thegenomic sequence of GANAB Intron 23 is shown in SEQ ID NO: 57. The DNAsequence for patient PA3 GANAB Intron 23 (containing SNP V) is shown inSEQ ID NO: 58. E) MAN1A1 SNP IX and X (Exon 13 3′ UTR): The DNA sequencefor the genomic sequence of MAN1A1 Exon 13 3′ UTR is shown in SEQ ID NO:59. The DNA sequence for patient PAJ MAN1A1 Exon 13 3′ UTR (containingSNP IX and X) is shown in SEQ ID NO: 60. F) MGAT1 SNP VI (Exon 2 3′UTR): The DNA sequence for the genomic sequence of MGAT1 Exon 2 3′ UTRis shown in SEQ ID NO: 61. The DNA sequence for patient PA7 MGAT1 Exon 23′ UTR (containing SNP VI) is shown in SEQ ID NO: 62. G) MGAT5 SNP I(Exon 1 5′ UTR): The DNA sequence for the genomic sequence of MGAT5 ExonI 5′ UTR is shown in SEQ ID NO: 63. The DNA sequence for patient PA2MGAT5 Exon 1 5′ UTR (containing SNP I) is shown in SEQ ID NO: 64. H)MGAT5 SNP II (Intron 5): The DNA sequence for the genomic sequence ofMGAT5 Intron 5 is shown in SEQ ID NO: 65. The DNA sequence for patientPA7 MGAT5 Intron 5 (containing SNP II) is shown in SEQ ID NO: 66. I)MGAT5 SNP V (Intron 13): The DNA sequence for the genomic sequence ofMGAT5 Intron 13 is shown in SEQ ID NO: 67. The DNA sequence for patientPA1 MGAT5 Intron 13 (containing SNP V) is shown in SEQ ID NO: 68. Thesequences referred to above as “exon” or “intron” are used forconvenience and may refer to more or less than the full length exon orintron sequences.

FIG. 12. Inhibition of Mgat5 glycan expression by Glucosidase I/II withCST. A) Mouse T cells purified by negative selection (R&D Systems) wereleft unstimulated or stimulated with Anti-CD3 for 4 days in the presenceor absence of CST and analyzed by FACS for L-PHA staining. Shown isgated on CD4⁺ population. B) Jurkat T cells cultured with increasingconcentrations of CST were left untreated or co-cultured with GlcNAc (20mM)+Uridine for 3 days and analyzed by FACS for L-PHA staining.

FIG. 13. β1,6GlcNAc-branched N-glycans titrate TCR sensitivity and aredifferentially expressed in inbred strains of mice. (A, B) FACS analysisof resting (A) and 3 day stimulated CD4+ T-cells from PL/J (A,B) and129/Sv (A) mice using L-PHA, a plant lectin that specifically bindsβ1,6GlcNAc-branched N-glycans (FIG. 7A). Error bars are standard errorof triplicate staining. MFI=Mean Fluorescence Intensity. (C) PurifiedCD3+ PL/J T-cells were labeled with CFSE, stimulated for 72 hrs andanalyzed by FACS. Plots shown are gated on CD4+ cells. (D,F) PurifiedPL/J CD3+ T-cells were incubated at 37° C. with anti-CD3 antibody-coatedbeads for various times, lysed and western blotted (WB). (E) Splenocytesfrom the indicated inbred mouse strains were stained with L-PHA andanti-CD4, anti-CD8 or anti-B220 antibodies and analyzed by FACS. Shownis relative expression normalized to wild type 129/Sv cells. n=number ofmice. Error bars are standard error and p values are by theKruskal-Wallis Anova test. (G, H) CD3+ T-cells (G,H) or splenocytes (G)were lysed and used to assess enzyme activity (G) or to isolate mRNA forcDNA synthesis and analysis by quantitative real time-PCR (2) (H). Shownin H is relative expression normalized to 129/Sv cells and represents 3mice done in triplicate. Error bars are standard error.

FIG. 14. Metabolic regulation of β1,6GlcNAc-branched N-glycanexpression, T-cell function and EAE by the hexosamine pathway. (A-C) Theindicated monosaccharides and metabolites were cultured with JurkatT-cells for 3 days, stained with L-PHA-FITC and analyzed by FACS (A,C)or lysed and analyzed by MS/MS mass spectroscopy for sugar-nucleotideexpression (B). Lines with symbols ▪, ▴, ▾ refer to altered glucoseconcentration in the culture media as indicated; all others were grownin 10 mM glucose. Error bars in C are standard error of triplicatestaining. (D,E) Wildtype PL/J CD3+ T-cells unlabelled (D) or labeledwith CFSE (E) were stimulated with anti-CD3 antibody in the presence ofswainsonine and/or GlcNAc as indicated for 3 days and analyzed for L-PHA(I)) or CFSE (E) staining. Shown are gated on CD4+ cells. (F-H)Splenocytes isolated from wild type PL/J mice 11 days after immunizationwith MBP+CFA were re-stimulated in vitro with MBP for 4 days in thepresence (green) or absence (red) of GlcNAc (40 mM), stained withL-PHA-FITC and LEA-FITC (F), tested for IFN-γ and IL-6 production inharvested supernatant (G) and 3.6 million CD3+ cells were injected intonaïve Mgat5+/−PL/J mice (n=7 for each condition) and scored for signs ofEAE daily for 30 days (H). Shown in G is standard error of duplicatevalues. P values for disease incidence and mean clinical score weredetermined by Fisher's exact test and Mann-Whitney test, respectively.

FIG. 15. Demyelinating pathology in PL/J mice, β1,6GlcNAc-branchedN-glycan expression in MS patient T-cells and functional analysis ofMGAT1 SNP IV/V. (A-C) Paraffin embedded sections from spinal cord (A),brainstem (B) and spinal roots (C) from clinically affected PL/J micewere stained with Luxol Fast Blue (B,C) or Haematoxylin & Eosin (A).Green arrows point to large naked axons. (D) Freshly isolated PBMCs fromCaucasian MS patients and Caucasian controls were left unstimulated orstimulated with anti-CD3 antibody or a mixture of myelin antigens for 48hrs and then analyzed for L-PHA staining levels. Fold change wascalculated by comparing the MFI of blasting cells vs. non-stimulatedcells as defined by side vs. forward scatter. Shown is gated on CD4+. Pvalues are by the Mann-Whitney t test. (E) MGAT1 enzyme activity inPBMCs containing MGAT1 SNP IV/V (n=3), MGAT1 SNP IV (n=1) or the commonallele (n=1). (F-H) Lec1 cells transiently transfected with humanpCMV-MGAT1 with or without SNP IV/V were lysed to assess enzyme activity(F), isolate mRNA for cDNA synthesis and quantitative real time PCR (G)or L-PHA FACS analysis in the absence (H,J) or presence of GlcNAc (J).L-PHA MFI was determined on L-PHA+ population. F and G were normalizedfor transfection efficiency as determined by L-PHA+ vs. L-PHA-cells.Result in H was the same in 8 separate transient transfections. Allerror bars are standard error of duplicate (F) or triplicate and greater(E, G, H, J) values. (I) Ordinary Differential Equation (ODE) model ofmedial Golgi enzyme reactions showing predicted alterations in β1,6GlcNAc branched N-glycans with two-fold changes in MGAT1 activity.

FIG. 16. MALDI-TOF mass spectroscopy of N-Glycans from mice and humancontrols and MS patients. (A) N-glycan processing pathway demonstratingGolgi intermediates and associated monoisotopic mass (permethylated,Na+) on MALDI-TOF mass spectroscopy. Glycan species were grouped basedon enzymatic function and MALDI-TOF profiles of CHO cells defective atvarious steps in the N-glycan pathway (FIG. 21). Structures in group Awere found as 4, 5 and 6 hexose fragments as observed in Glucosidase I(GI) deficient CHO (Lec23) cells (see FIG. 21) and are likely producedby endo-mannosidase activity at the arrowheads. Mass species 1662, 1907and 2153 were not reliably detected. (B, C) N-glycans obtained frommouse CD3+ T-cells and MS and control PBMCs were analyzed by MALDI-TOFmass spectroscopy. Relative intensity of structures A-G were obtained byadding the intensity of structures from each group and dividing by thecombined intensity of mature bi-antennary (2071) and tri-antennary(2520) glycans.

FIG. 17. Intermediate T-cell activation thresholds in Mgat5+/−129/Sv Tcells and computational model of N-glycan processing. (A) Purified CD3+129/Sv T cells were labeled with CFSE, stimulated for 72 hrs asindicated and analyzed by FACS. Plots shown are gated on CD4+ cells. (B)β1,6GlcNAc-branched N-glycan as a fraction of total cellular N-glycanscomputed using experimentally determined enzyme activities of Mgat1,2and 5 for the three indicated mouse strains. Last three bars aresimulations beginning with 129/Sv parameters and substitution of PL/JMgat1, Mgat2 and Mgat5 enzyme activities individually.

FIG. 18. Proximal N-glycan processing regulates β1,6GlcNAc-branchedN-glycan expression. (A-B) Jurkat T cells were incubated with thealkaloids castanospermine (CST), deoxymannojirimycin (DMN) and/orswainsonine (SW) for 3 days to inhibit proximal N-glycan processing (seeFIG. 7A) and analyzed by L-PHA flow cytometry. Shown is the relativechange in staining compared to untreated. Error bars are S.E.M fortriplicate staining. (C) mRNA isolated from Jurkat T cells at rest andstimulated with anti-CD3 antibody for the indicated doses and times wasreverse transcribed into cDNA and analyzed by Taqman quantitativeRT-PCR. Change in mRNA expression of the indicated genes following TCRstimulation is normalized to the resting state. (D,E) Mouse CD3+ T cells(D) or Jurkat T cells (E) were cultured with anti-CD3 in the presence ofminimal doses of CST, DMN and SW as indicated for 3 days and analyzed byFACS for L-PHA staining. Shown is gated on CD4+ population. (F) MouseCD3+ T cells were left unstained or stained with CFSE and stimulatedwith anti-CD3 antibody in the presence or absence of the indicated dosesof SW for 5 days. Shown is the LPHA MFI for non-CFSE labeled cells (leftpanel) and the percentage increase in the number of proliferating CD4+ Tcells relative to cells stimulated with 62.5 ng/ml anti-CD3 (rightpanel).

FIG. 19. Metabolic regulation of β1,6GlcNAc-branched N-glycans and TCRsensitivity by the Hexosamine pathway (A-D) The indicatedmonosaccharides, metabolites and glycosidase inhibitors were culturedwith Jurkat T cells for 3 days, stained with L-PHA or LEA, a lectinspecific for poly-N-acetyllactosamine, and analyzed by FACS. Green, blueand red lines refer to altered glucose concentration in the culturemedia as indicated; all others were grown in 10 mM glucose. Error barsare standard error of triplicate staining. (E) Jurkat T cells weretreated with the indicated concentrations of DMN or CST in the presenceor absence of GlcNAc (20 mM) and Uridine (10 mM) for 3 days and analyzedby FACS for L-PHA staining. (F) Wild-type C57/BL6 CD3+ T cells labeledwith CFSE were stimulated with anti-CD3 antibody in the presence orabsence of swainsonine and/or GlcNAc as indicated for 3 days andanalyzed by FACS.

FIG. 20. Dystonic posturing and pathological/physiological demyelinationin PL/J mice. (A) Clinically affected mice were observed to havedystonic posturing of the tail, hind limbs and/or axial skeleton. (B-H)Paraffin-embedded sections were stained with Haematoxylin & Eosin(C-E,H) or Luxol Fast Blue (B,F-G). B-D shows spinal cord demyelination,gliosis and neuronophagia. E shows axonal swelling in spinal cordsurrounded by otherwise normal appearing white matter. F showsmulti-focal myelin degeneration of spinal roots. G shows neuronal bodieswith central chromatolysis in the spinal cord. H shows spinal root withswollen axons. (I-J) PL/J mice underwent needle electromyography (EMG)and nerve conduction studies (NCS) for assessment of F waves and Hresponses, a clinical physiological test for spinal root demyelination.I shows example of the positive sharp waves observed. J shows thefrequency of delayed or absent F and H responses as assessed by NCS inmice with normal and abnormal needle EMG. (K) Frequency of spinal root(PNS) and CNS pathology in Mgat5+/+ (n=9), Mgat5+/− (n=10) and Mgat5−/−(n=17) PL/J mice (p=0.048, chi square for CNS).

FIG. 21. N-glycan profiling of CHO cells with defects in N-glycanprocessing. (A-F) CHO cells untreated or treated with DMN (mannosidase Iinhibition) or SW (mannosidase II inhibition) along with LecR mutant CHOcells Lec23 (Glucosidase I deficient), Lec1 (MGAT1 deficient) and/orLEC4 (MGAT5 deficient) were stained with ConA (binds high mannoseN-glycans), or L-PHA and analyzed by FACS (A) or total cellular lysateswere used to obtain N-glycans by PNGaseF digestion for MALDI-TOF massspectroscopy (B-F). MALDI-TOF spectra B-F are representative oftriplicate samples. Letters in red refer to N-glycan structures in FIG.16A. Lec23 cells demonstrated a large and specific increase in hexoseoligomers of 4, 5 and 6 residues in all three samples, consistent withGolgi endo-mannosidase activity. (G) CHO and Lec1 cells were mixed inthe indicated proportions and lysed. PNGaseF derived N-glycans wereanalyzed by MALDI-TOF and the relative intensity of C and G ions as aproportion of the total N-glycan intensities are plotted versus mixtureratios. (H) PBMC from MS patients PA2 and PA6 and Caucasian controlswere thawed and compared directly by L-PHA flow cytometry.

FIG. 22. Single nucleotide polymorphisms in genes controlling N-glycanprocessing in MS patients and controls. MS patients with alteredN-glycan processing are shown in red next to the genes controlling thesteps with accumulated N-glycan intermediates by MALDI-TOF. Sequencingof genomic DNA on PCR products from PA1-7,J for each exon identified 14previously unknown SNPs (red), which were confirmed by sequencing inboth directions in at least two separate PCR amplifications, as well as24 previously known SNPs (blue, NCBI SNP database). Exon and introns arenot to scale. MS and Control refer to the ratio of chromosomes +SNP and−SNP as determined by sequencing and/or allelic discrimination.Genotyping of GCS1, GANAB SNP IV and MAN1A1 SNP III by sequencingidentified GCS1, GANAB SNP III and MAN1A1 SNP IV. Green boxes refer tocorrelation (cSNP) with the MALDI-TOF profile in FIG. 21 and allelicfrequency ≦5% in control samples. Grey background defines all SNPs withallelic frequency ≦5% in control samples, which were confirmed by anallelic discrimination assay that is independent of Taq error (excludingMAN1A1 IV and GCS1, GANAB SNP V which required sequencing to genotype).Het. refers to predicted heterozygosity as defined in the NCBI SNPdatabase. SNPs that appeared linked are grouped together except MGAT1SNP I and SNP IV, which were observed together in a small sample size.MAN1A2 and MAN1C1 were not targeted for sequencing as the former doesnot map to the 18 MS chromosomal regions defined in Table 4 and thelatter is not significantly expressed in T cells. Sequences for thepreviously unknown SNPs (red) are in FIG. 23. The known SNPs (blue) areGCS1: II-rs1063588, IIIrs2268416; GCS1, GANAB: I-rs2957121,III-rs1063445, IV-rs10897289, VI-rs11231166; MAN1A1: II-rs4946409,111-1295388, V-rs6915947, VI-rs195092, VII-rs9481891, VIII-rs2072890,IX-rs2142887, X-rs3756943, XI-rs18513744, XII-rs3798602, XIIIrs1042800,XV-rs1046226. MGAT1: II-rs3733751, IV-rs7726357, V-rs2070924 andrs2070925, VI-rs634501. MGAT5: III-rs3214771, IV-rs3748900, V-rs2289465.

FIG. 23. Rare SNPs in GCS1, GCS1, GANAB, MAN1A1, MGAT1 and MGAT5associated with MALDI-TOF Glycan profile. A-Q). Sequences are shown forthe indicated SNPs with small, unlabeled boxes defining coding SNPs,arrow heads defining UTR and intronic SNPs and exon/intron junctions asindicated. A) GCS1 SNP 1 (Exon 1): The DNA sequence for the genomicsequence of GCS1 Exon I is shown in SEQ ID NO: 69. The correspondingamino acid sequence is shown in SEQ ID NO: 70. The DNA sequence forpatient PA3 GCS1 Exon 1 (containing SNP I) is shown in SEQ ID NO: 71.The corresponding amino acid sequence is shown in SEQ ID NO: 72. B)GANAB SNP II (Intron 6): The DNA sequence for the genomic sequence ofGANAB Intron 6 is shown in SEQ ID NO: 73. The DNA sequence for patientsPA4 and PA6 GANAB Intron 6 (containing SNP II) is shown in SEQ ID NO:74. C) GANAB SNP III & V (Exon 10 and Intron 10): The DNA sequence forthe genomic sequence of GANAB Exon 10 and Intron 10 (first line) isshown in SEQ ID NO: 75. The DNA sequence corresponding to the secondline containing the [A/G] SNP IV is shown in SEQ ID NO: 76. The DNAsequence corresponding to the third and fourth lines containing theArg→Cys SNPIII (Exon 10) is shown in SEQ ID NO: 77. D) GANAB SNP V (Exon11): The DNA sequence for the genomic sequence of GANAB Exon 11 is shownin SEQ ID NO: 78. The corresponding amino acid sequence is shown in SEQID NO: 79. The DNA sequence for patient PA3 GANAB Exon 11 (containingSNP V) is shown in SEQ ID NO: 80. The corresponding amino acid sequenceis unchanged and is shown in SEQ ID NO: 79. E) GANAB SNP VI and VII(Exons 20 and 21): The DNA sequence for the genomic sequence of GANABExons 20 and 21 is shown in SEQ ID NO: 81. The DNA sequence for patientPAJ GANAB Exons 20 and 21 (containing SNP VI) is shown in SEQ ID NO: 82.The DNA sequence for patient PA1 GANAB Exons 20 and 21 (containing SNPVII) is shown in SEQ ID NO: 83. F) GANAB SNP VIII (Intron 23): The DNAsequence for the genomic sequence of GANAB Intron 23 is shown in SEQ IDNO: 84. The DNA sequence for patient PA3 GANAB Intron 23 (containing SNPVIII) is shown in SEQ ID NO: 85. G) MAN1A1 SNP I (Exon 1 5′ UTR) and SNPII (Intron I): The DNA sequence for the genomic sequence of MAN1A1 Exon1 and Intron 1 is shown in SEQ ID NO: 86. The DNA sequence for patientPA2 MAN1A1 Exon 1 (containing SNP I) and Intron 1 (containing SNP II) isshown in SEQ ID NO: 87. H) MAN1A1 SNP IV (Exon 2 5′ UTR): The DNAsequence for the genomic sequence of MAN1A1 Exon 2 5′ UTR is shown inSEQ ID NO: 88. The DNA sequence containing SNP IV in MAN1A1 Exon 2 5′UTRis shown in SEQ ID NO: 89. I) MAN1A1 SNP XIII and XIV (Exon 13 3′ UTR):The DNA sequence for the genomic sequence of MAN1A1 Exon 13 3′ UTR isshown in SEQ ID NO: 90. The DNA sequence for patient PAJ MAN1A1 Exon 133′ UTR (containing SNP XIII and XIV) is shown in SEQ ID NO: 91. J) MGAT1SNP I (Promoter and Exon 1 5′ UTR): The DNA sequence for the genomicsequence of MGAT1 Promoter and Exon 1 5′ UTR is shown in SEQ ID NO: 92.The DNA sequence for patient PAJ MGAT1 Promoter and Exon 1 5′ UTR(containing SNP I) is shown in SEQ ID NO: 93. The DNA sequence forpatient PA7 MGAT1 Promoter and Exon 1 5′ UTR (containing SNP I) is shownin SEQ ID NO: 94. K) MGAT1 SNP II (Exon 2 5′ UTR) and SNP III (Exon2-synonymous): The DNA sequence for the genomic sequence of MGAT1 Exon 25′ UTR and Exon 2 is shown in SEQ ID NO: 95. The DNA sequence forpatients PA2 and PA4 MGAT1 Exon 2 5′ UTR and Exon 2 (containing SNP II)is shown in SEQ ID NO: 96. The DNA sequence for patient PA1 MGAT1 Exon 25′ UTR and Exon 2 (containing SNP III) is shown in SEQ ID NO: 97. L)MGAT1 SNP V (Exon 2): The DNA sequence for the genomic sequence of MGAT1Exon 2 is shown in SEQ ID NO: 98. The corresponding amino acid sequenceis shown in SEQ ID NO: 99. The DNA sequence for patients PA1, PA2, PA4and PA7 MGAT1 Exon 2 (containing SNP V) is shown in SEQ ID NO: 100. Thecorresponding amino acid sequence is unchanged and is shown in SEQ IDNO: 99. M) MGAT1 SNP VII (Exon 2 3′ UTR): The DNA sequence for thegenomic sequence of MGAT1 Exon 2 3′ UTR is shown in SEQ ID NO: 101. TheDNA sequence for patient PA7 MGAT1 Exon 2 3′ UTR (containing SNP VII) isshown in SEQ ID NO: 102. N) MGAT5 SNP 1 (Exon 1 5′ UTR): The DNAsequence for the genomic sequence of MGAT5 Exon 1 5′ UTR is shown in SEQID NO: 103. The DNA sequence for patient PA2 MGAT5 Exon 1 5′ UTR(containing SNP I) is shown in SEQ ID NO: 104. O) MGAT5 SNP II (Intron5): The DNA sequence for the genomic sequence of MGAT5 Intron 5 is shownin SEQ ID NO: 105. The DNA sequence for patient PA7 MGAT5 Intron 5(containing SNP II) is shown in SEQ ID NO: 106. P) MGAT5 SNP V (Intron13): The DNA sequence for the genomic sequence of MGAT5 Intron 13 isshown in SEQ ID NO: 107. The DNA sequence for patient PA1 MGAT5 Intron13 (containing SNP V) is shown in SEQ ID NO: 108. Q) MGAT5 SNP VI(Intron 14): The DNA sequence for the genomic sequence of MGAT5 Intron14 is shown in SEQ ID NO: 109. The DNA sequence for patient PAJ MGAT5Intron 14 (containing SNP VI) is shown in SEQ ID NO: 110. The sequencesreferred to above as “exon” or “intron” are used for convenience and mayrefer to more or less than the full length exon or intron sequences.

FIG. 24 Enhanced expression of β1,6GlcNAc-branched N-glycan by VitaminD3 and supplements to the hexosamine pathway. (A-D) The indicatedmonosaccharides and/or metabolites were cultured with Jurkat T-cells for3 days, stained with L-PHA-FITC and analyzed by FACS.

DETAILED DESCRIPTION OF THE INVENTION

Glossary

In accordance with the present invention there may be employedconventional techniques of molecular biology and polynucleotidechemistry within the skill of the art. Such techniques are explainedfully in the literature. See for example, Sambrook et al, MolecularCloning: A Laboratory Manual, Third Edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Polynucleotide Hybridization B. D.Hames & S. J. Higgins eds. (1985); Transcription and Translation B. D.Hames & S. J. Higgins eds (1984); Oligonucleotide Synthesis (M. J. Gait,ed., 1984); the series, Methods in Enzymology (Academic Press, Inc.);the series Current Protocols in Human Genetics (Dracopoli et al., eds.,1984 with quarterly updates, John Wiley & Sons, Inc.); Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif.; Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2004); Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Fundamental Methods SpringerLab Manual, Springer-Verlag (Berlin Heidelberg New York); and Atlas andParks (Eds.), The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla., all of which are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following definitionssupplement those in the art and are directed to the present applicationand are not to be imputed to any related or unrelated case. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice for testing of the invention, particularmaterials and methods are described herein.

Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

The terms “administering” or “administration” refers to the process bywhich a therapeutically effective amount of compounds or a compositioncontemplated herein are delivered to a patient for preventive ortreatment purposes. Compounds and compositions are administered inaccordance with good medical practices taking into account the patient'sclinical condition, the site and method of administration, dosage,patient age, sex, body weight, and other factors known to physicians.

An “agonist” is used in its broadest sense. Agonist can include anyagent that results in activation, enhancement or alteration of thepresence or expression of a component of an N-glycan or hexosaminepathway (e.g., Mgat5), including polynucleotides encoding the component,in particular mRNA or DNA, or results in activation, enhancement oralteration of the presence or expression of N-glycans (e.g. Mgat5modified glycans and/or polylactosamine modified glycans). Agonists mayinclude proteins, peptides, polynucleotides, carbohydrates, or any othermolecules that provide the desired activation, enhancement, oralteration. An agonist may activate, enhance or alter a pathway forsynthesis of a sugar donor. The stimulation may be direct, or indirect,or by a competitive or non-competitive mechanism.

“Allele” refers to different sequence variants found at differentpolymorphic regions. The sequence variants may be single or multiplebase changes, including without limitation insertions, deletions, orsubstitutions, or may be a variable number of sequence repeats.

“Allele frequency” refers to the frequency that a given allele appearsin a population.

An “antibody” is a multi-subunit protein produced by a mammalianorganism in response to an antigen challenge. Antibodies include but arenot limited to monoclonal antibodies, polyclonal antibodies, antibodyfragments (e.g. a Fab or (Fab)₂ fragments), antibody heavy chains,humanized antibodies, antibody light chains, genetically engineeredsingle chain F_(v) molecules (Ladner et al, U.S. Pat. No. 4,946,778),recombinantly produced binding partners, chimeric antibodies, forexample, antibodies which contain the binding specificity of murineantibodies, but in which the remaining portions are of human origin, orderivatives, such as enzyme conjugates or labeled derivatives.

Antibodies including monoclonal and polyclonal antibodies, fragments andchimeras, may be prepared using methods known to those skilled in theart. Isolated native or recombinant polypeptides may be utilized toprepare antibodies. See, for example, Kohler et al. (1975) Nature256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote etal. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) MolCell Biol 62:109-120 for the preparation of monoclonal antibodies; Huseet al. (1989) Science 246:1275-1281 for the preparation of monoclonalFab fragments; and, Pound (1998) Immunochemical Protocols, Humana Press,Totowa, N.J. for the preparation of phagemid or B-lymphocyteimmunoglobulin libraries to identify antibodies.

Antibodies specific for a polypeptide may also be obtained fromscientific or commercial sources. In an embodiment of the invention,antibodies are reactive against a polypeptide if they bind with a K_(a)of greater than or equal to 10⁻⁷ M. Binding partners may be constructedutilizing recombinant DNA techniques to incorporate the variable regionsof a gene which encodes a specifically binding antibody (See Bird etal., Science 242:423-426, 1988).

“Arrays”, “micro arrays” or “DNA chips” refer to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. A microarray may be prepared and usedaccording to the methods described in U.S. Pat. No. 5,837,832, Chee etal., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al.(1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.Natl. Acad. Sci. 93: 10614-10619) or produced by the methods describedby Brown et al., U.S. Pat. No. 5,807,522. Any number of probes, such asallele-specific oligonucleotides, may be used in an array, wherein eachprobe or pair of probes corresponds to a different polymorphism (e.g.,SNP position). Oligonucleotides can be synthesized at designated areason a substrate using a light-directed chemical process. Hybridizationassays based on arrays rely on differences in hybridization stability ofoligonucleotide probes to perfectly matched and mismatched targetsequence variants. Each array can contain thousands to millions ofindividual synthetic DNA probes arranged in a grid-like pattern, eachcorresponding to a particular SNP position or allelic variant.

A “disease” refers to any disease which can be treated by the preventiveand therapeutic methods and compositions of the invention includingwithout limitation conditions or diseases associated or related toglycan dysregulation. In aspects of the invention the disease isassociated or related to alterations (e.g. decrease or reduction) in acomponent of a N-glycan pathway or hexosamine pathway (e.g., Mgat5) orN-glycans (e.g., Mgat5 modified glycans and/or polylactosamine modifiedglycans). Examples of such diseases include autoimmune diseases such asinsulin-dependent diabetes mellitus, autoimmune demyelinating diseases(e.g., multiple sclerosis, chronic inflammatory demyelinatingpolyneuropathy), rheumatoid arthritis, myasthenia gravis, systemic lupuserythematosus, autoimmune hemolytic anemia, glomerulonephritis, enhanceddelayed type hypersensitivity, scleroderma, Sjogren's syndrome,Wegener's granulomatosis, polymyalgia rheumatica, temporalarteritis/giant cell arteritis, allergic conditions, hypersensitivity,Hashimoto's thyroiditis (underactive thyroid), Graves' disease(overactive thyroid), psoriasis, Celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, Addison's disease, primarybiliary sclerosis, Sclerosing cholangitis, autoimmune hepatitis, andRaynaud's phenomenon.

Autoimmune demyelinating diseases may be categorized into the diseaseswherein the demyelination occurs in the central nervous system (CNS) andthe diseases wherein the demyelination occurs in the peripheral nervoussystem (PNS). Examples of diseases associated with the demyelination inthe central nervous system are acute disseminated encephalomyelitisincluding idiopathic acute disseminated encephalomyelitis, postinfectious acute disseminated encephalomyelitis, post vaccinal acutedisseminated encephalomyelitis and the like, multiple sclerosisincluding concentric sclerosis, neuromyelitis optica (Devic's disease),and the like. These diseases, and in particular multiple sclerosis, canundergo recurring remission and relapse and both the disease in theremission phase and the relapse phase are to be diagnosed, and treatedby the methods and compositions of the invention acting as a preventiveagent and a therapeutic agent, respectively. Diseases associated withdemyelination in the peripheral nerve system include without limitationchronic, inflammatory demyelinating polyradiculitis/polyneuropathy(CIDP) and the like, and acute, inflammatory, demyelinatingpolyradiculitis/polyneuropathy (i.e., Gullian Barre Syndrome) and thelike.

In certain aspects of the invention the methods and compositions areused in the treatment and prevention of diseases wherein thedemyelination occurs in the central nervous system.

In aspects of the invention the disease is a Th1-mediated(cell-mediated) autoimmune diseases including: multiple sclerosis (MS),rheumatoid arthritis (RA), autoimmune thyroiditis, and uveitis, inparticular MS.

The term “dysregulation” as used herein in conjunction with a componentof a cell refers to any effect that alters at least one of the activityand quantity of the component in the cell as compared to a cell notaffected by that dysregulation. For example, a dysregulation of Mgat5refers to a reduction in the amount of properly expressed Mgat5 glycansin a cell (e.g., less than 70% are expressed as compared to normal), areduction in expression or catalytic activity of Mgat5 and other enzymesin the N-glycan or hexosamine pathways upstream of Mgat5 (e.g., K_(M)increased by 20% or V_(MAX) decreased by 25%), and/or presence of Mgat5or upstream N-glycan or hexosamine pathway enzymes in an environmentthat is depleted in at least one substrate, or altered in pH.

“GANAB” refers to an AB isozyme of neutral alpha-glucosidase AB thathydrolyzes the terminal 1,3-alpha-D-glucosidic links in1,3-alpha-D-glucans, preferably the mammalian enzyme. Examples of GANABenzymes include human GANAB (GeneID 23193; Treml Ket al, Glycobiology.2000 May 10(5):493-502; SEQ ID NOs. 20-25), and mouse GNAB (AccessionNo. NM_008060; Arendt, C. W. and Ostergaard, H. L, J. Biol. Chem. 272(20), 13117-13125 (1997)). “GANAB” includes the wild type enzyme, orpart thereof, or a mutant, variant or homolog of such an enzyme. Wherethe context admits, the term refers to a polynucleotide or gene encodinga GANB enzyme including the coding region, non-coding region preceding(leader) and following coding regions, introns, and exons of a GANABsequence. In particular, the GANAB gene includes the promoter.

“Genetic predisposition”, “genetic susceptibility” and “susceptibility”all refer to the likelihood that a subject will develop a disease (e.g.an autoimmune disease). A subject with an increased susceptibility orpredisposition will be more likely than average to develop a diseasewhile a subject with a decreased predisposition will be less likely thanaverage to develop the disease. A genetic variant is associated with analtered susceptibility or predisposition if the allele frequency of thegenetic variant in a population with a disease or disorder varies fromits allele frequency in a control population without the disease ofdisorder or a wild type sequence by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 50%, 55%, 60% or 65%.

“Genetic variant” or “variant” refers to a specific genetic variantwhich is present at a particular genetic locus in at least oneindividual in a population and that differs from the wild type.

“GCS1” refers to glucosidase I, which is the first enzyme in theN-linked oligosaccharide processing pathway. GCS1 cleaves the distalalpha-1,2-linked glucose residue from the Glc(3)-Man(9)-GlcNAc(2)oligosaccharide precursor. The term preferably refers to the mammalianenzyme. Examples of GCS1 enzymes include human GCS1 (Gene ID: 7841;Accession NO. NM_006302; Kalz-Fuller B, et al, Eur J Biochem. 1995 Jul.15; 231(2):344-51. Erratum in: Eur J Biochem 1997 Nov. 1; 249(3):912;SEQ ID NOs. 18 and 19), and rat GCS1 (GeneID: 78947; AccessionNM_031749). “GCS1” includes the wild type enzyme, or part thereof, or amutant, variant or homolog of such an enzyme. Where the context admits,the term refers to a gene or polynucleotide encoding a GCS1 enzymeincluding the coding region, non-coding region preceding (leader) andfollowing coding regions, introns, and exons of a GCS1sequence. Inparticular, the GCS1 gene includes the promoter.

The term “genotype” refers to the identity of alleles present in anindividual or a sample. In the context of the present invention the termparticularly refers to the description of the polymorphic allelespresent in an individual or a sample. “Genotyping” a sample or anindividual for a polymorphic marker involves determining the specificallele or the specific nucleotide carried by an individual at apolymorphic marker.

The term “haplotype” refers to the combination of alleles on onechromosome. In the context of the present invention it may refer to acombination of polymorphisms found in an individual which may beassociated with a phenotype.

The “hexosamine pathway” refers to the pathway leading to the formationof UDP-N-acetylglucosamine (UDP-GlcNAc) from glucose. FIG. 7A shows aschematic diagram of the hexosamine pathway.

“MAN1A1” refers to a mannosidase, alpha, class 1A, member which is atype II transmembrane protein. This protein catalyzes the removal of 3distinct mannose residues from peptide-bound Man(9)-GlcNAc(2)oligosaccharides and belongs to family 47 of glycosyl hydrolases. Theterm preferably refers to the mammalian enzyme. Examples of GANABenzymes include human MAN1A11 (Gene ID: 4121; Accession No. NM_005907;Tremblay L O and Herscovics A Glycobiology. 1999 October; 9(10):1073-8;SEQ ID NOs. 26 and 27), and canine MAN1A1 (GeneID: 431698; Accession No.AY514736). “MAN1A1” includes the wild type enzyme, or part thereof, or amutant, variant or homolog of such an enzyme. Where the context admits,the term refers to a polynucleotide or gene encoding a MAN1A1 enzymeincluding the coding region, non-coding region preceding (leader) andfollowing coding regions, introns, and exons of a MAN1A1 sequence. Inparticular, the MAN1A1 gene includes the promoter.

“Mgat1” refers to UDP-N-acetylglucosamine: alpha-3-D-mannosidebeta-1,2-N-acetyl glucose aminyltransferase I which is a medial-Golgienzyme essential for the synthesis of hybrid and complex N-glycans. Theprotein shows typical features of a type II transmembrane protein. Theterm preferably refers to the mammalian enzyme. Examples of Mgat1enzymes include human Mgat1 (Gene ID: 4245; Accession NO. NM_002406;Kumar et al. Proc Natl Acad Sci USA. 1990 December; 87(24):9948-52; SEQID NOs. 28 and 29), and rat Mgat1 (GeneID: 81519; Accession No.NM_030861). “Mgat1” includes the wild type enzyme, or part thereof, or amutant, variant or homolog of such an enzyme. The term “MGAT1” refers toa gene or polynucleotide encoding an Mgat1 enzyme, including the codingregion, non-coding region preceding (leader) and following codingregions, introns, and exons of a MGAT1 sequence. In particular, theMGAT1 gene includes the promoter.

“Mgat2” refers to mannosyl (alpha-1,6-)-glycoproteinbeta-1,2-N-acetylglucosaminyltransferase. The protein is a Golgi enzymecatalyzing an essential step in the conversion of oligomannose tocomplex N-glycans. The enzyme has the typical glycosyltransferasedomains: a short N-terminal cytoplasmic domain, a hydrophobicnon-cleavable signal-anchor domain, and a C-terminal catalytic domain.The term preferably refers to the mammalian enzyme. Examples of Mgat2enzymes include human Mgat2 (Gene ID: 4247; Accession Nos. NM_001015883,NM_002408, NP_001015883 and NP_002399; SEQ ID NOs.32, 33, and 34), andrat Mgat2 (GeneID: 94273 Accession Nos. NM_053604 and NP_446056).“Mgat2” includes the wild type enzyme, or part thereof, or a mutant,variant or homolog of such an enzyme. The term “MGAT2” refers to a geneor polynucleotide encoding an Mgat2 enzyme, including the coding region,non-coding region preceding (leader) and following coding regions,introns, and exons of a MGAT2 sequence. In particular, the MGAT2 geneincludes the promoter.

“Mgat5” refers to β1,6N-acetylglucosaminyltransferase V enzymes,preferably mammalian enzymes that catalyze the addition ofN-acetylglucosamine in beta 1-6 linkage to the alpha-linked mannose ofbiantennary N-linked oligosaccharides. Examples of Mgat5 enzymes arefound on the ExPASy proteomics server as Enzyme: 2.4.1.155, and includehuman Mgat5 (Saito et al, 1994; gb:d17716, sw:q09328; SEQ. ID. Nos. 30and 31), and rat Mgat5 (Shoreibah et al 1993, J. Biol. Chem. 268:15381-15385; gb114284, sw:q08834;). “Mgat5” includes the wild typeenzyme, or part thereof, or a mutant, variant or homolog of such anenzyme. The term “MGAT5” refers to a gene or polynucleotide encoding anMgat5 enzyme, including the coding region, non-coding region preceding(leader) and following coding regions, introns, and exons of a MGAT5sequence. In particular, the MGAT5 gene includes the promoter. A“promoter” is a regulatory sequence of DNA that is involved in thebinding of RNA polymerase to initiate transcription of a gene and isconsidered part of the corresponding gene.

“Mgat5 modified glycan” refers to a GlcNAβ1,6Manα1,6-branched N-glycanstructure. The glycans are produced by Mgat5 which catalyzes theaddition of β1,6GlcNAc to N-glycan intermediates found on newlysynthesized glycoproteins transiting the medial Golgi (Cummings, R. D.,et al., J. Biol. Chem., 257: 13421-13427 (1982). The glycans areelongated in trans-Golgi to produce tri (2, 2, 6) and tetra (2, 4, 2, 6)antennary N-glycans. A Mgat5 modified glycan may be substituted with forexample polylactosamine (i.e. it may be a polylactosamine modifiedglycan). A Mgat5 modified glycan may be part of or covalently linked toa cell surface glycoprotein, including a glycoprotein of the T cellreceptor complex.

“N-glycans” refers to asparagine (N)-linked oligosaccharides. AllN-linked oligosaccharides are linked to the amide N in the sidechain ofaspargine in the consensus sequence Asn-Xaa-Ser/Thr, where Xaa can beany amino acid besides Pro and Asp. N-glycans can be subdivided intothree distinct groups called ‘high mannose type’, ‘hybrid type’, and‘complex type’, with the common pentasaccharidecore—Manp(alpha1,6)-(Manp(alpha1,3))-Manp(beta1,4)-GlcpNAc(beta1,4)-GlcpNAc(beta1,N)-Asn—occuringin all three groups. N-glycans include Mgat5 modified glycans andpolylactosamine modified glycans.

“N-glycan pathway” refers to the pathway by which N-glycans areprocessed or synthesized. FIG. 7A shows a schematic diagram of theN-glycan pathway.

As used herein “nutraceutically acceptable derivative” refers to aderivative or substitute for the stated chemical species that operatesin a similar manner to produce the intended effect, and is structurallysimilar and physiologically compatible. Examples of substitutes includewithout limitation salts, esters, hydrates, or complexes of the statedchemical. The substitute could also be a precursor or prodrug to thestated chemical, which subsequently undergoes a reaction in vivo toyield the stated chemical or a substitute thereof.

The term “pharmaceutically acceptable carrier, excipient, or vehicle”refers to a medium which does not interfere with the effectiveness oractivity of an active ingredient and which is not toxic to the hosts towhich it is administered. A carrier, excipient, or vehicle includesdiluents, binders, adhesives, lubricants, disintegrates, bulking agents,wetting or emulsifying agents, pH buffering agents, and miscellaneousmaterials such as absorhants that may be needed in order to prepare aparticular composition. Examples of carriers etc. include but are notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The use of such media and agents for an activesubstance is well known in the art.

“Polylactosamine modified glycan” refers to specific glycan structurescomprising N-acetyllactosamine (Galβ1,4GlcNAc) and polymeric forms ofN-acetyllactosamine, also known as poly N-acetyllactosamine orpolylactosamine (Cummings, R. D. and Kornfeld, S. J. Biol. Chem., 259:6253-6260 (19840). Preferably the polylactosamine modified glycan is anMgat5 modified glycan substituted with poly N-acetyllactosamine. Apolylactosamine modified glycan may be part of or covalently linked to acell surface glycoprotein, including a glycoprotein of the T cellreceptor complex.

“Polymorphism” or“polymorphism site” refers to a set of genetic variantsat a particular genetic locus among individuals in a population. A“single nucleotide polymorphism” (SNP) occurs at a polymorphic siteoccupied by a single nucleotide which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele. In the context of the presentinvention the term refers to a set of genetic variants in genetic lociassociated with the N-glycan pathway or hexosamine pathway co-localizedin chromosomal regions associated with a disease disclosed herein (e.g.,an autoimmune disease, in particular MS or rheumatoid arthritis), orgenetic loci associated with a disease disclosed herein or apredisposition to a disease disclosed herein. In aspects of theinvention the term refers to genetic variants of a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and/or MGAT5 gene.

Polymorphism sites may correspond to one or more polymorphisms within oroutside the promoter region of a gene of the N-glycan pathway orhexosamine pathway, in particular a GCS1, GANAB, MAN1A1, MGAT1, MGAT2,and MGAT5 gene. Other polymorphism sites, including polymorphisms linkedto these polymorphisms, may be determined using methods known in the artand/or disclosed herein. It will be noted that the numericaldesignations of the positions of the polymorphisms within a sequence aresequence specific and the same numerical positions may be assigneddifferent numerical designations depending on the way in which thesequence is numbered and the sequence selected. In addition, sequencevariations within a population, including insertions or deletions maychange the relative position of the polymorphism and subsequently thenumerical designations of particular nucleotides at and around apolymorphism. Sequences for specific polymorphisms are in FIGS. 11, 22,and 23, and SEQ. ID NOs.:5, 6, 7, 8, 9 and 35-47.

The terms “polynucleotide” and “oligonucleotide” refer tosingle-stranded or double-stranded nucleotide polymers comprised of morethan two nucleotide subunits covalently joined together. The nucleotidesmay comprise ribonucleotides, deoxyribonucleotides, and/or any otherN-glycoside of a purine or pyrimidine base, or modified purine orpyrimidine bases, non-standard or derivatized base moieties (see forexample, U.S. Pat. Nos. 6,001,611, 5,955,589, 5,844,063, 5,789,562,5,750,3343, 5,728,525, and 5,679,785), or any combination thereof. Thesugar groups of the nucleotide subunits may also comprise modifiedderivatives of ribose or deoxyribose, (e.g. o-methyl ribose). Subunitsmay be joined by phosphodiester linkages, phosphorothioate linkages,methyl phosphonate linkages or by other linkages, including rare ornon-naturally occurring linkages that do not interfere withhybridization. An oligonucleotide may have uncommon nucleotides ornon-nucleotide subunits.

The term “primer” refers to an oligonucleotide that has a hybridizationspecificity sufficient for the initiation of an enzymatic polymerizationunder predetermined conditions in an amplification reaction, asequencing method, a reverse transcription method, and similar reactionsand methods. For example, a primer can be a single-strandedoligonucleotide capable of acting as a point of initiation oftemplate-directed DNA synthesis under suitable conditions (e.g., in thepresence of four different nucleoside triphosphates and an agent forpolymerization, such as DNA or RNA polymerase or reverse transcriptase)in an appropriate buffer and at a suitable temperature. The length of aprimer will depend on its intended use but typically ranges from 15 to30 nucleotides. A primer need not reflect the exact sequence of thetemplate but it must be sufficiently complementary to hybridize with atemplate.

A “probe” refers to a polynucleotide capable of binding in abase-specific manner to a complementary strand of polynucleotide, suchas a complementary strand of polynucleotide to be identified in a sampleunder predetermined conditions, for example in an amplificationtechnique such as a 5′-nuclease reaction, a hybridization-dependentdetection method (e.g. Southern or Northern blot), and the like.

Hybridizations with nucleic acids or probes can be generally performedunder “stringent conditions”. Stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example,hybridizations may be performed at a salt concentration of no more than1M and a temperature of at least 25° C. In methods of the invention forallele-specific probe hybridizations, conditions of 5×SPPE (750 mM NaCL,50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. canbe used. Another example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.However, one skilled in the art could readily substitute othercompositions of equal suitability. Examples of moderate to lowstringency hybridization conditions are also well known in the art.

Probes may be immobilized on a solid support by covalent bonding,absorption, hydrophobic and/or electrostatic interaction, directsynthesis on a solid support, and the like. The probes may be labeledwith labels such as radioactive isotopes, enzymes, in particular enzymescapable of acting on a chromogenic, fluorescent or luminescent substrate(e.g. peroxidase or alkaline phosphatase), chromophoric chemicalcompounds, acridinium esters (see U.S. Pat. No. 5,185,439), substrates,cofactors, inhibitors, magnetic particles, chromogenic, fluorigenic orluminescent compounds, analogues of nucleotide bases, and ligands (e.g.,biotin). Examples of fluorescent compounds include fluorescein,carboxyfluorescein, tetrachloro fluorescein, hexachlorofluorescein, Cy3,Cy3.5, Cy5, tetramethylrhodamine, rhodamine and its derivatives (e.g.carboxy-X-rhodamine), and Texas Red. Examples of luminescent compoundsinclude luciferin, and 2,3-dihydrophthalazinediones (e.g. luminol).Examples of radioactive isotopes include ³H, ³⁵S, ³²P ¹²⁵I, ⁵⁷Co, and¹⁴C. Many labels are commercially available and can be used in thecontext of the present invention.

Probes and primers can be modified with chemical groups to enhance theirperformance or facilitate the characterization of hybridization oramplification products. In an aspect of the invention, the probes orprimers have modified backbones (e.g., phosphorothioate ormethylphosphonate groups) which render the oligonucleotides resistant tothe nucleolytic activity of certain polymerases or to nucleases.Non-nucleotide linkers (e.g. EP No. 0313219) that do not interfere withhybridization or elongation of the primer can also be incorporated inthe polynucleotide chain. A 3′ end of an amplification primer or probemay be blocked to prevent initiation of DNA synthesis (see WO 94/03472),or the 5′ end may be modified so that it is resistant to the5′exonuclease activity present in some polymerases.

Oligonucleotides that are primer or probe sequences may comprise DNA,RNA, or polynucleotide analogs including uncharged polynucleotideanalogs such as peptide polynucleotides (PNAs) (see PCT PublishedApplication No. WO92/20702; Nielsen et al, Science 254, 1497-1500,1991), morpholino analogs (see U.S. Pat. Nos. 5,185,444, 5,034,506, and5,142,047), and N3′-P5′-phosphoamidate (PA) analogs (see for example,U.S. Pat. No. 6,169,170).

Polynucleotides and oligonucleotides may be prepared using methods knownin the art, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as conventional purification methods. Forexample, polynucleotides and oligonucleotides can be synthesized usingnucleotide phosphoramidite chemistry, in particular using instrumentsavailable from Applied Biosystems, Inc (Foster City, Calif.), DuPont(Wilmington, Det) or Milligen (Bedford, Mass.).

When desirable, polynucleotides and oligonucleotides may be labeledusing methods known in the art (see U.S. Pat. Nos. 5,464,746; 5,424,414;and 4,948,882). Polynucleotides and oligonucleotides, including labeledor modified polynucleotides and oligonucleotides, can also be obtainedfrom commercial sources. For example, polynucleotides andoligonucleotides can be ordered from QIAGEN (http://oligos.qiagen.com),The Midland Certified Reagent Company (www.mcrc.com), and ExpressGen Inc(Chicago, Ill.).

The term “sample” and the like mean a material known or suspected ofexpressing or containing a component of a N-glycan or hexosamine pathway(in particular, an enzyme of a N-glycan or hexosamine pathway such asMgat5), N-glycans (e.g., Mgat5 modified glycans or polylactosaminemodified glycans), a polynucleotide comprising a disease-associatedpolymorphism (e.g., SNP-containing polynucleotide of the invention), apolypeptide variant of the invention, or a gene of the N-glycan pathwayor hexosamine pathway, in particular a GCS1, GANAB, MAN1A1, MGAT1,MGAT2, and/or MGAT5 gene.

A test sample can be used directly as obtained from the source orfollowing a pretreatment to modify the character of the sample. Thesample can be derived from any biological source, such as tissues,extracts, or cell cultures, including cells, cell lysates, andphysiological fluids, such as, for example, whole blood, plasma, serum,saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk,ascites fluid, synovial fluid, peritoneal fluid and the like. The samplecan be treated prior to use, such as preparing plasma from blood,diluting viscous fluids, and the like. Methods of treatment can involvefiltration, distillation, extraction, concentration, inactivation ofinterfering components, the addition of reagents, and the like.Polynucleotides may be isolated from samples and utilized in the methodsof the invention. Thus a test sample may be a polynucleotide sequencecorresponding to the sequence in the test sample, that is all or part ofthe region in the sample polynucleotide may first be amplified using aconventional technique such as PCR, before being analyzed for sequencevariation. A polynucleotide sample may comprise RNA, mRNA, DNA, cDNA,genomic DNA, and oligonucleotides, and may be double-stranded orsingle-stranded. The polynucleotides may be sense strand, the non-codingregions, and/or the antisense strand, and can include all or a portionof the coding sequence of the gene, and may further comprise additionalnon-coding regions such as introns, and non-coding sequences includingregulatory sequences (e.g. a promoter). A polynucleotide can be fused toa marker sequence, for example, a sequence that is used to purify thepolynucleotide.

In embodiments of the invention the sample is a mammalian sample,preferably human sample. In another embodiment the sample is aphysiological fluid.

The term “sequencing” refers to a method for determining the order ofnucleotides in a polynucleotide. Methods for sequencing polynucleotidesare well known in the art and include the Sanger method ofdideoxy-mediated chain termination (for example, see. Sanger et al.,Proc. Natl. Acad. Sci. 74:5463, 1977; “DNA Sequencing” in Sambrook etal. (eds), Molecular Cloning: A Laboratory Manual (Second Edition),Plainview, N.Y.: Cold Spring Harbor Laboratory Press (1989)); theMaxam-Gilbert chemical degradation of DNA (Maxam and Gilbert, MethodsEnzymol. 65:499 (1980); and “DNA Sequencing” in Sambrook et al., supra,1989); and automated methods, for example, mass spectrometry methods.

The terms “subject”, “individual”, or “patient” refer to an animalincluding a warm-blooded animal such as a mammal, which is afflictedwith or suspected of having or being pre-disposed to, or at risk ofdeveloping as disease disclosed herein especially an autoimmune disease,in particular MS or rheumatoid arthritis. Mammal includes withoutlimitation any members of the Mammalia. In general, the terms refer to ahuman. The terms also include animals bred for food, pets, or sports,including domestic animals such as horses, cows, sheep, poultry, fish,pigs, and goats, cats, dogs, zoo animals, apes (e.g. gorilla orchimpanzee), and rodents such as rats and mice. The methods herein foruse on subjects/individuals/patients contemplate prophylactic as well ascurative use. Typical subjects for treatment include persons susceptibleto, suffering from or that have suffered a disease discussed hereinespecially an autoimmune disease or related disease, in particular MS orrheumatoid arthritis.

“Synergistic” means a greater pharmacological or therapeutic effect withthe use of a multi-component composition or combination therapy (e.g.GlcNAc and uridine or uracil) than with the use of one of the compoundsalone. This synergistic effect can work through either similar ordifferent mechanisms or pathways of action. One advantage of acombination therapy with a synergistic effect is that standard dosagescan be used for a greater therapeutic effect than expected from theaddition of the effect of one or two compounds as the case may beadministered alone; or alternatively lower dosages or reduced frequencyof administration of the therapeutic compound(s) may be used to achievea better therapeutic effect.

“Therapeutically effective amount” relates to the amount or dose of anactive compound, composition, or combination therapy of the inventionthat will lead to one or more desired beneficial effects. Atherapeutically effective amount of a compound, composition, orpreparation can vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the substanceto elicit a desired response in the individual. Dosage regime may beadjusted to provide the optimum therapeutic response (e.g. beneficialeffects). For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

The terms “trait” and “phenotype”, used interchangeably herein, refer toany visible, detectable, or otherwise measurable property of an organismsuch as symptoms of, or susceptibility to a disease disclosed herein(e.g., an autoimmune disease). Generally, the terms are used herein torefer to symptoms, or susceptibility to a disease disclosed herein(e.g., an autoimmune disease), or to an individual's response to anagent acting on a disease, or to symptoms of, or susceptibility to sideeffects to an agent acting on a disease.

The term “treating” refers to reversing, alleviating, or inhibiting theprogress of a disease, or one or more symptoms of such disease, to whichsuch term applies. Depending on the condition of the subject, the termalso refers to preventing a disease, and includes preventing the onsetof a disease, or preventing the symptoms associated with a disease. Atreatment may be either performed in an acute or chronic way. The termalso refers to reducing the severity of a disease or symptoms associatedwith such disease prior to affliction with the disease. Such preventionor reduction of the severity of a disease prior to affliction refers toadministration of a compound or composition of the present invention toa subject that is not at the time of administration afflicted with thedisease. “Preventing” also refers to preventing the recurrence of adisease, the relapse of a disease after remission, or of one or moresymptoms associated with such disease. The terms “treatment” and“therapeutically,” refer to the act of treating, as “treating” isdefined above.

DESCRIPTION OF EMBODIMENTS

Animal Model

The invention provides a nonhuman animal characterized by lacking one orboth Mgat5 gene alleles at least in its somatic cells and displayingpathology of an autoimmune demyelinating disease, in particular a CNSdemyelinating disease, more particularly MS. In aspects of the inventionthe incidence, severity, and/or mortality of disease in the nonhumananimal are characterized by an inverse correlation with Mgat5 products.

The invention also provides a nonhuman animal that is usable as ananimal model presenting CNS pathology or PNS pathology similar tochronic MS plaques or CIDP. In an aspect, the invention provides anonhuman animal that is usable as an animal model presenting a clinicalcondition typical of primary progressive multiple sclerosis (PPMS) andsecondary progressive multiple sclerosis (SPMS) in screening a remedyfor demyelinating autoimmune diseases, more particularly multiplesclerosis. In an aspect, the invention provides a nonhuman animal thatis usable as an animal model presenting a clinical condition typical ofprimary progressive multiple sclerosis (PPMS) and secondary progressivemultiple sclerosis (SPMS) in screening a remedy to prevent theneurodegeneration in MS.

Another aspect of the invention provides a nonhuman animal presentingpathological conditions of human Chronic Inflammatory DemyelinatingPolyneuropathy (CIDP).

In a further aspect, the present invention provides a transgenicknockout rodent, in particular a mouse or rat, lacking one or both Mgat5gene alleles at least in its somatic cells and displaying pathology of aCNS autoimmune demyelinating disease.

In an embodiment a transgenic knockout rodent is provided (e.g. PL/Jmice) which are hypomophic for Mgat5-modified N-glycans).

In embodiments of the present invention, nonhuman animals are providedwith somatic and germ cells having a functional disruption of one, orpreferably both, alleles of an endogenous Mgat5 gene. Accordingly, theinvention provides viable nonhuman animals having a mutated or deletedMgat5 gene and thus lacking or substantially lacking Mgat5 activity.Animals of the invention can display a chronic and slowly progressiveclinical course without relapses or recovery and involuntary movementsin a Mgat5 gene dose dependent manner. The involuntary movements caninclude tremor and/or focal dystonic posturing of the tail, hindlimbsand/or spine, and/or paroxysmal episodes of dystonia common in patientswith MS.

In some embodiments, the nonhuman animals display pathology similar tochronic MS plaques which can be characterized by mononuclear cellsadmixed with myelin debris centered around blood vessels, gliosis,neuronophagia, axonal swelling (spheroids) and axonal degeneration.Axonal pathology may also be observed in otherwise normal appearing CNSwhite matter. PNS pathology can be characterized by multi-focal spinalroot demyelination with naked and swollen axons. Neuronal bodies withprominent central chromatolysis may be observed in the spinal cord,consistent with anterograde reaction to peripheral damage. Myokymia,positive sharp waves and delayed spinal root nerve condition velocitymay be typical of physiologic spinal root demeylination and the humanPNS autoimmune demyelinating disease Chronic Inflammatory DemyelinatingPolyneuropathy (CIDP).

A nonhuman animal of the invention is exemplified by mice lacking one orboth alleles of Mgat5 as more particularly described in the Examplesherein.

The present invention further relates to a method for generatingnonhuman animals of the invention. Any method for generating knockoutanimals is contemplated by the present invention. Such methods includepronuclear microinjection, retrovirus mediated gene transfer into germlines, gene targeting in embryonic stem cells, electroporation ofembryos, and sperm-mediated gene transfer. In certain embodiments, thepresent invention provides methods for generating a transgenic animalcomprising crossing a first Mgat5 knockout EAE resistant animal and asecond EAE sensitive animal. In some rodent embodiments (e.g. mice) thefirst animal is a 129/Sv knockout animal and the second animal is a PL/Janimal strain. A method for generating a knockout animal can furthercomprise backcrossing the progeny onto an EAE sensitive animal.

The invention provides a method of using a nonhuman or transgenic animalof the invention as a model animal of an autoimmune demyelinatingdisease, in particular multiple sclerosis, comprising measuring theextent of presentation of characteristics similar to an autoimmunedemyelinating disease, in particular multiple sclerosis, moreparticularly PPMS and SPMS. The characteristics may include the displayof clinical condition and pathology described herein, and/or the amountof Mgat5 modified glycans on T cells obtained from the animals.

The invention provides a transgenic nonhuman animal assay system whichprovides a model system for testing a compound that reduces or inhibitspathology associated with a condition or disease described herein,comprising:

-   -   (a) administering the compound to a transgenic nonhuman animal        of the invention; and    -   (b) determining whether said compound reduces or inhibits the        pathology in the transgenic non-human animal relative to a        transgenic non-human animal of step (a) which has not been        administered the agent.

The compound may be useful in the treatment and prophylaxis of diseasesdiscussed herein. The compounds may also be incorporated in apharmaceutical composition, and may optionally comprise apharmaceutically acceptable carrier, vehicle or excipient.

The present invention also provides methods of screening a test compoundcomprising exposing a nonhuman animal of the invention to the testcompound; and determining a response of the animal to the test compound.In certain embodiments, a change in response compared to an animal notexposed to the test compound indicates a response to the compound. Inother embodiments, the animals (cells, tissues, or organs) are examineddirectly and compared to a wild-type animal or animal not exposed to thetest compound.

In embodiments of the invention, the compound tested is a candidatecompound for treatment or prevention of a condition described herein, inparticular an autoimmune demyelinating disease, in particular multiplesclerosis. In other embodiments, the compound is a known compound forthe treatment or prevention of a condition described herein, inparticular an autoimmune demyelinating disease, in particular multiplesclerosis.

The present invention provides a method of conducting a drug discoverybusiness comprising:

-   -   (a) providing methods for screening compounds as described        herein;    -   (b) conducting therapeutic profiling of compounds identified in        step (a), or further analogs thereof, for efficacy and toxicity        in animals; and    -   (c) formulating a pharmaceutical preparation including one or        more compounds identified in step (b) as having an acceptable        therapeutic profile.

In certain embodiments, the subject method can also include a step ofestablishing a distribution system for distributing the pharmaceuticalpreparation for sale, and may optionally include establishing a salesgroup for marketing the pharmaceutical preparation.

Yet another aspect of the invention provides a method of conducting adrug discovery business comprising:

-   -   (a) providing methods for screening compounds described herein;    -   (b) (optionally) conducting therapeutic profiling of compounds        identified in step (a) for efficacy and toxicity in animals; and    -   (c) licensing, to a third party, the rights for further drug        development and/or sales for agents identified in step (a), or        analogs thereof.

The method may further comprise the steps of preparing a quantity of acompound and/or preparing a pharmaceutical composition comprising thecompound.

Polynucleotides

The present invention relates to isolated polynucleotides oroligonucleotides that contain one or more SNPs of a gene of a N-glycanor hexosamine pathway. The present invention further provides isolatedpolynucleotides that encode the variant protein. Such polynucleotides oroligonucleotides will consist of, consist essentially of, or compriseone or more SNPs of the present invention. The polynucleotides can haveadditional nucleic acid residues, such as nucleic acid residues that arenaturally associated with it or heterologous nucleotide sequences.

An isolated SNP-containing polynucleotide comprises a SNP of the presentinvention separated from other nucleic acid present in the naturalsource of the nucleic acid. Generally, the isolated SNP-containingpolynucleotide, as used herein, will be comprised of one or more SNPpositions disclosed by the present invention with flanking nucleotidesequence on either side of the SNP positions. A flanking sequence maycomprise about 300 bases, 100 bases, 50 bases, 30 bases, 15 bases, 10bases, or 4 bases on either side of a SNP position for detectionreagents or as long as the entire protein encoding sequence if it is tobe used to produce a protein containing a coding variants. Apolynucleotide or oligonucleotide is generally isolated from remote andunimportant flanking sequences and is of appropriate length such that itcan be subjected to the specific manipulations or uses described herein,including recombinant expression, preparation of probes and primers forthe SNP position, and other uses specific to the SNP-containingpolynucleotides or oligonucleotides.

An isolated polynucleotide, such as a cDNA comprising a SNP of thepresent invention is generally substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized. Apolynucleotide comprising a SNP can be fused to other coding orregulatory sequences and still be considered isolated. For example, arecombinant DNA contained in a vector or maintained in heterologous hostcells is considered isolated. In vivo or in vitro RNA transcripts of anisolated SNP-containing DNA molecules is considered an isolated RNA. Anisolated SNP-containing polynucleotide can be in the form of RNA, suchas mRNA, or in the form of DNA, including cDNA and genomic DNA obtainedby cloning or produced by chemical synthetic procedures or by acombination thereof. Isolated SNP-containing polynucleotides can bedouble-stranded or single-stranded; single-stranded polynucleotides canbe the coding strand (sense strand) or the non-coding strand (anti-sensestrand).

Polynucleotides that hybridize under stringent conditions to theisolated SNP-containing polynucleotides are also contemplated. Forexample, hybridization conditions may be selected so that nucleotidesequences encoding a peptide at least 60-70%, 80%, 90% or morehomologous to each other typically remain hybridized to each other.

An aspect of the invention provides polynucleotides, for example,polynucleotides comprising one or more novel polymorphisms in a geneassociated with a disease disclosed herein (e.g. an autoimmune disease,in particular) and/or oligonucleotides useful for detecting suchpolymorphisms. Accordingly, one embodiment of the invention is anisolated polynucleotide molecule comprising a portion of a gene, itscomplement, and/or a variant thereof. In particular aspects, the variantcomprises a polymorphism identified herein. More particularly, thevariant comprises at least one of the polymorphisms identified herein tobe associated with a disease disclosed herein (e.g. an autoimmunedisease, in particular MS). In a further embodiment, the polynucleotidemolecule comprises or consists of a primer and/or a probe specific to atleast one of the polymorphisms identified in a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and/or MGAT5 gene (e.g., those identified herein to beassociated with disease disclosed herein, in particular an autoimmunedisease, more particularly MS).

In embodiments of the invention a SNP-containing polynucleotide is apolynucleotide shown in FIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7,8, 9, and 35-47.

The invention also provides a vector comprising a SNP-containingpolynucleotide. Vectors can be for maintenance (cloning vectors) or forexpression (expression vectors) of the SNP-containing polynucleotidesThe term “vector” refers to a vehicle, preferably a nucleic acidmolecule that can transport a SNP-containing polynucleotide. When thevector is a polynucleotide, the SNP-containing nucleic acidpolynucleotides are covalently linked to the vector nucleic acid. Suchvectors include plasmids, single or double stranded phages, single ordouble stranded RNA or DNA viral vectors, or artificial chromosomes,such as a BAC, PAC, YAC, OR MAC. A vector can function in procaryotic oreukaryotic host cells or in both (shuttle vectors).

A vector can be introduced into an appropriate host cell for propagationor expression using well-known techniques. Therefore, the invention alsorelates to recombinant host cells containing the vectors describedherein. Recombinant host cells are prepared by introducing the vectorsinto the cells by techniques know to a person of ordinary skill in theart. These include, but are not limited to, calcium phosphatetransfection, DEAE-dextran-mediated transfection, cationiclipid-mediated transfection, electroporation, transduction infection,lipofection, and other techniques such as those found in Sambrook, etal. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989). Host cells can include prokaryotic cells, lowereukaryotic cells such as yeast, other eukaryotic cells such as insectcells, and higher eukaryotic cells such as mammalian cells.

Polypeptide Variants/Antibodies

A polymorphic SNP-containing polynucleotide may encode a variant of agene product (polypeptide). Therefore, the present invention providesisolated variant polypeptides that comprise, consist of or consistessentially of one or more variant amino acids encoded by anonsynonymous nucleotide substitution at one or more of the SNPpositions disclosed herein; also referred to as variant amino acids,polypeptides, or proteins encoded by SNPs disclosed herein. A variantpolypeptide includes, but is not limited to deletions, additions andsubstitutions in the amino acid sequence of the polypeptide caused bySNPs of the present invention. One class of substitutions is conservedamino acid substitutions where a given amino acid is substituted foranother amino acid of like characteristics. Examples of conservativesubstitutions are replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.[See Bowie et al., Science 247:1306-1310 (1990) concerning amino acidchanges are likely to be phenotypically silent.]

A variant polypeptide may be fully functional or can lack function inone or more activities, e.g. enzymatic activity. A fully functionalvariant generally contains only a conservative variation or a variationin non-critical residues or in non-critical regions. A functionalvariant may also contain a substitution of similar amino acids thatresults in no change or an insignificant change in function.Alternatively, substitutions may affect function to some degree. Anon-functional variant generally contains one or more non-conservativeamino acid substitution, deletion, insertion, inversion, or truncationin a critical residue or critical region.

A variant polypeptide is typically “isolated” or “purified”, that is, itsubstantially free of cellular material or free of chemical precursorsor other chemicals. A variant polypeptide can be purified to homogeneityor other degrees of purity. The level of purification will be based onthe intended use and should allow for the desired function of thevariant polypeptide, even if in the presence of considerable amounts ofother components.

An isolated variant polypeptide can be purified from cells thatnaturally express it or from cells that have been altered to express it(recombinant). An isolated variant polypeptide can also be synthesizedusing known protein synthesis methods. By way of example, apolynucleotide containing SNP encoding a variant polypeptide can becloned into an expression vector, the expression vector introduced intoa host cell and the variant polypeptide expressed in the host cell. Thevariant polypeptide can then be isolated from the cells using standardprotein purification techniques.

Accordingly, the present invention provides variant polypeptides thatconsist of amino acid sequences that contain one or more of the aminoacid polymorphisms encoded by a SNP of the invention. A polypeptideconsists of an amino acid sequence when the amino acid sequence is thefinal amino acid sequence of the polypeptide.

The present invention further provides variant polypeptides that consistessentially of amino acid sequences that contain one or more of theamino acid polymorphisms encoded by a SNP of the invention. Apolypeptide consists essentially of an amino acid sequence when such anamino acid sequence is present with only a few additional amino acidresidues in the final polypeptide.

The present invention further provides variant polypeptides that arecomprised of amino acid sequences that contain one or more of the aminoacid polymorphisms encoded by a SNP of the invention. A polypeptidecomprises an amino acid sequence when the amino acid sequence is atleast part of the final amino acid sequence of the polypeptide. Apolypeptide can be only the variant polypeptide or have additional aminoacid molecules, such as amino acid residues (contiguous encodedsequence) that, are naturally associated with it or heterologous aminoacid residues/peptide sequences. Such a variant polypeptides can have afew additional amino acid residues or can comprise several hundred ormore additional amino acids.

Variant polypeptides can be attached to heterologous sequences to formchimeric or fusion proteins. Such chimeric and fusion proteins comprisea variant polypeptide operatively linked to a heterologous polypeptidehaving an amino acid sequence not substantially homologous to thevariant polypeptide. “Operatively linked” means that the variantpolypeptide and the heterologous protein are fused in-frame. Aheterologous polypeptide can be fused to the N-terminus or C-terminus ofthe variant polypeptide. A chimeric or fusion protein may be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different protein sequences can be ligated together in-frameusing conventional techniques, or a fusion gene can be synthesized byconventional techniques including automated DNA synthesizers. PCRamplification of gene fragments can also be carried out using anchorprimers which provide complementary overhangs between two consecutivegene fragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see Ausubel et al., Current Protocolsin Molecular Biology, 1992). In addition, expression vectors that encodea fusion moiety (e.g., a GST protein) are commercially available. Apolynucleotide encoding a variant polypeptide can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thevariant polypeptide.

A variant polypeptides may contain amino acids other than the 20naturally occurring amino acids. In addition, many amino acids,including the terminal amino acids, in a variant polypeptide may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inpolypeptides are well known to those of skill in the art. Thus, avariant polypeptide includes derivatives or analogs in which asubstituted amino acid residue is not one encoded by the genetic code. Avariant polypeptide may also comprise an amino acid with a substituentgroup fused with another compound, such as a compound to increase thehalf-life of the polypeptide (for example, polyethylene glycol), orfused with a leader or secretory sequence or a sequence for purificationof the mature polypeptide or a pro-protein sequence.

Known polypeptide modifications include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Fragments of a variant polypeptide containing one or more amino acidpolymorphisms and polypeptides and peptides that comprise and consist ofsuch fragments are also with the scope of the present invention. Thefragments of the invention are not to be construed as encompassingfragments that maybe disclosed publicly prior to the present invention.A fragment may comprise at least 8 or more contiguous amino acidresidues from a variant protein, wherein at least one residue is avariant amino acid encoded by a nonsynonymous nucleotide substitution ata SNP position provided by the present invention. A fragment can bechosen based on the ability to retain one or more of the biologicalactivities of the variant polypeptide or on the ability to perform afunction, e.g. catalytic activity. Preferably the fragments arebiologically active fragments.

The invention also provides antibodies that selectively bind to thevariant polypeptides of the present invention as well as fragmentsthereof. Such antibodies may be used to quantitatively or qualitativelydetect the variant polypeptides. Generally, an antibody selectivelybinds a target variant polypeptide when it binds the variant polypeptideand does not significantly bind to non-variant polypeptides, i.e., theantibody does not bind to wild-type, or previously disclosedpolypeptides that do not contain a variant amino acid encoded by anonsynonymous nucleotide substitution at a SNP position disclosedherein.

Antibodies of variant polypeptides can be prepared from any region ofthe variant polypeptide provided that the region contains a variantamino acid encoded by a nonsynonymous nucleotide substitution at a SNPposition. However, preferred regions will also include those involved infunction/activity and/or protein/binding partner interaction.

An antibody that binds a variant polypeptide can be detected by couplingthe antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials.

Diagnostic Applications

The invention contemplates methods of, and products for, diagnosing andmonitoring a disease disclosed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease such as multiple sclerosis, in a sample from asubject, comprising assaying for an alteration or change in N-glycans(e.g., Mgat5 modified glycans, or polylactosamine modified glycans),and/or in components (in particular polypeptides (e.g., enzymes) andgenes encoding the polypeptides, enzyme substrates, cofactors, andproducts) of a N-glycan pathway or hexosamine pathway, in a sample fromthe subject compared to a standard.

N-glycans (e.g., Mgat5 modified glycans, or polylactosamine modifiedglycans) and components (in particular polypeptides (e.g., enzymes) andgenes encoding the polypeptides, enzyme substrates, cofactors, andproducts) of an N-glycan pathway or hexosamine pathway can be assayedusing a variety of methods known to the skilled artisan. For example,enzyme assays can be used to assay for glucosidase I (GCS1),glucosidase, alpha, neutral AB (GANAB), glucosidase II, mannosidase I(MI), Mannosidase, alpha, class 1A, member 1 (MAN1A1), mannosidase II(MII/MIIx), MGAT1, MGAT2, and MGAT5 activity or expression. Mgat5modified glycans and polylactosamine modified glycans may be assayedusing substances that hind to the glycans. Substances that bind to theglycans may be antibodies or lectins. For example, leukoagglutinin(L-PHA) is a tetravalent plant lectin that binds specifically to Mgat5modified glycans and tomato lectin (LEA), which is a plant lectin thathinds N-acetylpolylactosamine. In an aspect, expression levels of acomponent of an N-glycan or hexosamine pathway are quantitated usingbinding agents such as antibodies or their fragments (e.g., via ELISA,western blot, Flourescent Activated Cell Sorting (FACS), etc.) orlectins (L-PHA and LEA binding assays). Components of an N-glycan orhexosamine pathway may also be quantitated by determination of enzymatickinetics of the polypeptides (e.g., K_(M), kcat, etc.) or by massspectroscopy. In particular, MALDI-TOF mass spectrometry can be used toprofile the N-glycans present in patient sample for diagnosis ofdefective N-glycan processing leading to Mgat5 modified N-glycans.Similarly, it is also contemplated that a patient sample may be analyzedfor the presence or quantity of a substrate, product, and/or cofactorthat is required in an enzyme that is part of the pathway underinvestigation. For example, the quantity of Mgat5 product or its sugarnucleotide substrate UDP-GlcNAc and upstream metabolites may bedetermined in patients that are diagnosed for MS.

Accordingly in aspects of the invention, processes and kits fordetermining the identity of target N-glycans by mass spectrometry areprovided. The processes include the steps of determining the molecularmass of target N-glycans by mass spectrometry, and then comparing themass to a standard, whereby the identity of the N-glycans can beascertained. Identity includes, but is not limited to, identifying thetype of N-glycans or identifying a change in N-glycans. Selection of thestandard will be determined as a function of the information desired.

One process for determining the identity of a target N-glycan includesthe steps of a) obtaining a target N-glycan; b) determining themolecular mass of the target N-glycan by mass spectrometry, and c)comparing the molecular mass of the target N-glycan with the molecularmass of a corresponding known N-glylcan. By comparing the molecular massof the target with a known N-glycan, the identity of the target N-glycancan be ascertained. As disclosed herein, N-glycans can be isolated froma cell or tissue obtained from a subject such as a human. N-glycans canbe isolated from PBMC's of a subject by methods known in the art, forexample, by digestion with an enzyme such as trypsin followed bytreatment with PNGaseF.

In an aspect of the invention, a method is provided for screening for oridentifying a subject having or predisposed to a disease disclosedherein comprising:

-   -   a) obtaining a sample containing N-glycans from the subject;    -   b) determining the molecular mass of the N-glycans by mass        spectrometry;    -   c) comparing the molecular mass of the N-glycans with the        molecular mass of corresponding known N-glycans, thereby        determining the identity of the N-glycans wherein the N-glycans        are markers for the disease.

The process is performed using a mass spectrometric analysis, includingfor example, matrix assisted laser desorption ionization (MALDI),continuous or pulsed electrospray ionization, ionspray, thermospray, ormassive cluster impact mass spectrometry and a detection format such aslinear time-of-flight (TOF), reflectron time-of-flight, singlequadruple, multiple quadruple, single magnetic sector, multiple magneticsector, Fourier transform ion cyclotron resonance, ion trap, andcombinations thereof such as MALDI-TOF spectrometry, preferablyMALDI-TOF.

In an embodiment, the invention features a method of diagnosing, orassessing the prognosis of multiple sclerosis in a subject. The methodincludes providing a test sample from a subject and detecting in thetest sample Mgat5 and/or N-glycans (e.g., Mgat5 modified glycans and/orpolylactosamine modified glycans). The levels of Mgat5 and N-glycans inthe test sample are compared to a standard or control sample, which isderived from one or more individuals who have multiple sclerosissymptoms and have a known multiple sclerosis status, or from anindividual or individuals who do not show multiple sclerosis symptoms.MS status can include, for example, exacerbations, attacks, remissions,benign, moderate, malignant and stable stages of the disease.

In an embodiment, the invention features a method of diagnosing, orassessing the prognosis of rheumatoid arthritis in a subject. The methodincludes providing a test sample from a subject and detecting in thetest sample Mgat2 and/or N-glycans. The levels of Mgat2 and N-glycans inthe test sample are compared to a standard or control sample, which isderived from one or more individuals who have rheumatoid arthritissymptoms, or from an individual or individuals who do not showrheumatoid arthritis symptoms.

A standard may correspond to levels quantitated for another sample or anearlier sample from the subject, or levels quantitated for a controlsample. Levels for control samples from healthy subjects, differentstages or types of condition, may be established by prospective and/orretrospective statistical studies. Diagnosis may be made by a finding ofstatistically different levels of detected N-glycans (e.g., Mgat5modified glycans, or polylactosamine modified glycans) and components(e.g., polypeptides or genes) of a N-glycan pathway or hexosaminepathway associated with a disease disclosed herein (e.g, an autoimmunedisease, in particular autoimmune demyelinating disease, moreparticularly multiple sclerosis), compared to a control sample orprevious levels quantitated for the same subject. In certain diagnosticand monitoring applications of the invention, incidence, severity, andmortality of the condition are inversely correlated with Mgat5.

Certain aspects of the invention stem from the observation that at leastone polymorphism [e.g. single nucleotide polymorphism (SNP)] in a geneof a N-glycan pathway or hexosamine pathway, which gene co-localized inchromosomal regions associated with a disease disclosed herein (e.g.autoimmune disease), is correlated with an individual's risk for thedisease. The contribution or association of particular polymorphismswith disease phenotypes enables the polymorphisms to be used to developsuperior diagnostic tests that are capable of identifying individualswho express a detectable trait (e.g., a disease disclosed herein) as theresult of a specific genotype, or individuals whose genotype places themat risk of developing a detectable trait at a subsequent time.

Therefore, aspects of the invention provide methods for detecting anindividual's increased or decreased risk for a disease disclosed herein(e.g., an autoimmune disease). Still further embodiments providemethods, kits, reagents and arrays useful for detecting an individual'srisk for a disease disclosed herein (e.g., an autoimmune disease). Themethods of the invention may be useful to assess the predispositionand/or susceptibility of an individual to a disease disclosed herein(e.g., an autoimmune disease). A polymorphism may be particularlyrelevant in the development of a disease disclosed herein (e.g., anautoimmune disease), and thus the present invention may be used torecognize individuals who are particularly at risk of developing theseconditions.

In other aspects, the invention provides a method for screening patientscomprising obtaining from the patient sequence information for one ormore genes of the N-glycan pathway or hexosamine pathway, in particularGCS1, GANAB, MAN1A1, MGAT1, MGAT2, and MGAT5 gene sequence information,and determining the identity of one or more polymorphisms in the gene(s)that is indicative of a disease disclosed herein (e.g., an autoimmunedisease, in particular MS or rheumatoid arthritis). The patient may beat risk of developing a disease or have a disease.

A polymorphism may contribute to a phenotype (e.g. disease condition) ofan individual in different ways. Some polymorphisms occur within thecoding sequence of a polypeptide and contribute to the phenotype byaffecting the structure of the polypeptide or by influencingreplication, transcription, mRNA splicing, mRNA stability and/ortranslation. Other polymorphisms occur within noncoding regions and mayindirectly affect phenotype by influencing replication, transcription,mRNA splicing, mRNA stability, and/or translation. In addition, a singlepolymorphism may affect more than one trait, and a single polymorphismtrait may be affected by polymorphisms in different genes.

The diagnostic methods, reagents and kits of the invention may be basedon a single polymorphism or a group of polymorphisms. Combined detectionof a plurality of polymorphisms (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) generally will increase the probability of an accuratediagnosis. To further increase the accuracy of a diagnostic method ofthe invention, the polymorphism analysis may be combined with that ofother polymorphisms or other risk factors of a disease disclosed herein,including without limitation family history, diet, or environmentalfactors. Therefore, diagnostic methods of the invention are optionallycombined with known clinical methods, to diagnose a disease disclosedherein, in particular an autoimmune disease. Thus, the methodsoptionally include performing at least one clinical test for a diseasedisclosed herein, in particular an autoimmune disease, such as MagneticResonance Imaging of the brain.

A polymorphism detected using a method of the invention can be anypredisposing or protective polymorphism in a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and/or MGAT5 gene. In an embodiment of the invention, thepolymorphism can be any polymorphism identified as predisposing orprotective by methods taught herein. In another embodiment, thepolymorphism can be a single nucleotide polymorphism (SNP) in a gene ofthe N-glycan pathway or hexosamine pathway, in particular a GCS1, GANAB,MAN1A1, MGAT1, MGAT2, and/or MGAT5 gene. In a further embodiment,specific haplotypes in a N-glycan or hexosamine gene locus, inparticular a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 gene locusas well as specific combinations of, and interactions between SNPs atthese and other loci can be indicative of an increased or a decreasedrisk of a disease disclosed herein, (e.g., an autoimmune disease, inparticular MS or rheumatoid arthritis).

The invention provides a method of analyzing a polynucleotide from anindividual to determine which nucleotides are present at polymorphicsites within a gene of the N-glycan pathway or hexosamine pathway toidentify polymorphisms within the gene, wherein the gene is co-localizedin chromosomal regions associated with a disease disclosed herein (inparticular an autoimmune disease, more particularly MS or rheumatoidarthritis). The analysis can be performed on a plurality of individualswho are tested for the presence of the disease phenotype. The presenceor absence of a disease phenotype or propensity for developing a diseasestate can then be correlated with a base or set of bases present at thepolymorphic sites in the individual tested. Alternatively, thisdetermination step is performed in such a way as to determine theidentity of the polymorphisms.

In an aspect, the invention provides a method for detecting anindividual's increased or decreased risk for a disease disclosed herein(e.g., an autoimmune disease, in particular MS or rheumatoid arthritis)by detecting in a polynucleotide sample of the individual the presenceof at least one disease-associated polymorphism in a gene of a N-glycanpathway or hexosamine pathway co-localized in chromosomal regionsassociated with the disease, in particular a GCS1, GANAB, MAN1A1, MGAT1,MGAT2, and/or MGAT5 gene, wherein the presence of the at least onepolymorphism indicates the individual's increased or decreased risk forthe disease.

In a particular aspect of the invention, methods are used to determinean individual's risk for an autoimmune disease and related diseases. Inthe methods, the presence of at least one autoimmune disease-associatedpolymorphism in a polynucleotide sample of the individual is detected.In particular aspects, the autoimmune disease-associated polymorphism isa polymorphism in a gene of the N-glycan pathway or hexosamine pathwayco-localized in chromosomal regions associated with the disease, moreparticularly a polymorphism in a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, andMGAT5 gene. The presence of at least one polymorphism provides anindication of the individual's risk for an autoimmune disease. Theindividual's risk for an autoimmune disease can be either an increasedrisk or a decreased risk as compared to an individual without the atleast one polymorphism (e.g., an individual with a different allele atthat polymorphic site). Accordingly, the at least one polymorphism cancomprise a predisposing or a protective polymorphism in a gene of theN-glycan pathway or hexosamine pathway co-localized in chromosomalregions associated with the disease, in particular the polymorphism isin a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 gene.

In an embodiment, the invention provides a method for identifying apolymorphism in a gene sequence of a gene of the N-glycan pathway orhexosamine pathway, in particular a GCS1, GANAB, MAN1A1, MGAT1, MGAT2,and MGAT5 gene sequence, that correlates with a disease disclosedherein, in particular an autoimmune disease, more particularly MS orrheumatoid arthritis. The method may comprise obtaining gene sequenceinformation in respect to gene(s) of the N-glycan pathway or hexosaminepathway co-localized in chromosomal regions associated with the diseasefrom a group of patients with the disease, identifying a site of atleast one polymorphism in the gene(s), and determining genotypes at thesite for individual patients in the group. The genotypes may becorrelated with the disease severity, prognosis of the patient, ortreatments. In particular, the method is performed on a sufficientpopulation size to obtain a statistically significant correlation.

The invention provides a method for diagnosing or aiding in thediagnosis of disease disclosed herein (e.g., an autoimmune disease, inparticular MS or rheumatoid arthritis) in a subject comprising the stepsof determining the genetic profile of genes of N-glycan pathway orhexosamine pathway of the subject, in particular the genetic profile ofGCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 genes, therebydiagnosing or aiding in the diagnosis of the disease.

In an aspect the invention provides a method for diagnosing a geneticsusceptibility for a disease disclosed herein (e.g., an autoimmunedisease, in particular MS or rheumatoid arthritis) in a subjectcomprising obtaining a biological sample containing polynucleotides fromthe subject; and analyzing the polynucleotides to detect the presence orabsence of one or more polymorphism in a gene of a N-glycan pathway orhexosamine pathway of the subject, in particular polymorphisms in aGCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 gene, wherein apolymorphism is associated with a genetic predisposition for thedisease.

In another aspect, the invention provides a method for diagnosis of adisease disclosed herein (e.g. an autoimmune disease, in particular MSor rheumatoid arthritis) in a patient having, or at risk of developingthe disease comprising determining a genotype including one or morepolymorphism sites in a gene of a N-glycan pathway or hexosaminepathway, in particular a polymorphism in one or more of a GCS1, GANAB,MAN1A1, MGAT1, MGAT2, and MGAT5 gene, for the patient.

In another aspect the invention provides a method for the diagnosis of asingle nucleotide polymorphism (SNP) in a gene of a N-glycan pathway orhexosamine pathway of the subject, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and MGAT5 gene, in a human comprising determining thesequence of the polynucleotide of the human at the position of the SNP,and determining the status of the human by reference to a polymorphismin the gene.

In another aspect, the invention provides a method for detecting whethera subject is suffering from or is predisposed to developing a diseasedisclosed herein comprising detecting in a nucleic acid sample from asubject one or more alleles shown in FIGS. 11, 22, and 23, and SEQ IDNOs.:5, 6, 7, 8, 9, and 35-47, wherein the presence of the one or morealleles indicates that the subject is predisposed to the development ofthe disease or has the disease. The detecting step can be selected fromthe group consisting of: a) allele specific oligonucleotidehybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′nuclease digestion; f) single-stranded conformation polymorphism; g)allele specific hybridization; h) primer specific extension; and j)oligonucleotide ligation assay. Prior to or in conjunction with thedetection step a nucleic acid sample is subject to an amplificationstep.

In an aspect the invention provides a method of analyzing apolynucleotide comprising obtaining a polynucleotide from an individualand determining the base occupying position of a SNP as shown in FIGS.11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47.

In an aspect the invention provides a method of analyzing apolynucleotide comprising obtaining a polynucleotide from an individualand determining the base occupying position of a SNP nucleotide as shownin FIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47.

In an aspect of the invention, a method is provided for the diagnosis ofan autoimmune disease, in particular MS or rheumatoid arthritiscomprising: (a) obtaining sample polynucleotide from an individual; (b)detecting the presence or absence of one or more variant nucleotide asshown in FIGS. 11,22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47in a GCS1, GANAB, MAN1A1, MGAT1 and/or MGAT5 gene, respectively; and (c)determining the status of the individual by reference to thepolymorphism in the GCS1, GANAB, MAN1A1, MGAT1 and/or MGAT5 gene.

In another aspect, the invention provides a method for diagnosis of anautoimmune disease in a patient having or at risk of developing anautoimmune disease, in particular MS or rheumatoid arthritis, comprisingdetermining for the patient the genotype of one or more polymorphismsite in a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and MGAT5 gene as shown inFIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47. Themethod may further comprise comparing the genotype with known genotypeswhich are indicative of an autoimmune disease.

The present invention therefore provides a method of diagnosing anautoimmune disease, in particular MS or rheumatoid arthritis, ordetermining the presence or absence of a GCS1, GANAB, MAN1A1, MGAT1,MGAT2, and MGAT5 haplotype in a patient by obtaining material from thepatient comprising polynucleotides SNP-containing polynucleotides asshown in FIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47and determining the GCS1, GANAB, MAN1A1, MGAT1 and MGAT5 gene haplotype.

In a further aspect, the invention provides a method for diagnosis of apatient having or at risk of developing an autoimmune disease, inparticular MS or rheumatoid arthritis, comprising determining a genotypeincluding a polymorphism site in the GCS1, GANAB, MAN1A1, MGAT1, MGAT2,and/or MGAT5 gene for the patient, wherein the polymorphism is as shownin FIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47 or apolymorphism site linked thereto.

In another aspect, the invention provides a method for diagnosis ofrheumatoid arthritis in a patient having or at risk of developingrheumatoid arthritis, comprising determining for the patient thegenotype of one or more polymorphism site in a, MGAT1 gene, inparticular a MGAT1 SNP as shown in SEQ ID NO. 42, 43, and 44. The methodmay further comprise comparing the genotype with known genotypes whichare indicative of rheumatoid arthritis.

Genotyping may be determined at a combination of multiple polymorphismsites within the promoter region or outside the promoter region of agene(s) of the N-glycan pathway or hexosamine pathway co-localized inchromosomal regions associated with the disease, in particular a GCS1,GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 gene, or another gene of theN-glycan or hexosamine pathway.

In particular aspects, the presence of a polymorphism inherited from oneof an individual's parents provides an indication of the individual'srisk for a disease disclosed herein (e.g., an autoimmune disease, inparticular MS or rheumatoid arthritis). In other aspects, the presenceof the polymorphism inherited from both of the individual's parentsprovides an indication of the individual's risk for a disease disclosedherein (e.g., an autoimmune disease, in particular MS or rheumatoidarthritis).

In aspects of the invention methods for determining an individual's riskfor a disease disclosed herein in particular an autoimmune disease, moreparticularly MS or rheumatoid arthritis, are provided comprisingdetermining an individual's genotype at one or more polymorphic sites ina gene of the N-glycan pathway or hexosamine pathway, in particular aGCS1, GANAB, MAN1A1, MGAT1, MGAT2, and MGAT5 gene. A first genotype atthe one or more polymorphic sites is statistically associated with anincreased risk for an autoimmune disease as compared to a secondgenotype at the polymorphic site. Thus, for example, if the individual'sgenotype corresponds to the first genotype, the individual's risk for anautoimmune disease is greater than that of other individuals who havethe second genotype.

In an aspect, the invention relates to the identification ofindividual's at risk of a disease disclosed herein (e.g., an autoimmunedisease) by detecting allelic variation at one or more positions in agene associated with an N-glycan pathway or hexosamine pathway,optionally in combination with any other polymorphism in the gene thatis or becomes known.

In some aspects, the presence of a single allele of a particularpolymorphism is sufficient to indicate whether the individual's risk ofan autoimmune disease is increased or decreased. In other aspects, twocopies of an allele of a particular polymorphism must be present toindicate an increased or decreased risk of a disease disclosed herein,in particular an autoimmune disease. Determining the individual'sgenotype typically involves obtaining a polynucleotide sample from theindividual, and determining the individual's genotype by amplifying atleast a portion of a gene of the N-glycan or hexosamine pathway from thesample, the portion comprising one or more polymorphic sites. Suchamplification directly determines the genotype or facilitates detectionof one or more polymorphisms by an additional step. In one aspect, theindividual's genotype is determined by performing an allele-specificamplification or an allele-specific extension reaction. In anotheraspect, the individual's genotype is determined by sequencing at least aportion of the gene from the sample, the portion comprising at least onepolymorphic site. In yet another aspect, the individual's genotype isdetermined by hybridization of a polynucleotide probe, optionally afteramplification of at least a portion of the gene. In particular aspects,at least one polymorphic site consists of a single nucleotide position,and the sample is contacted with at least one sequence-specificoligonucleotide probe under stringent conditions. In an aspect, theprobe hybridizes under stringent conditions to polynucleotides in thesample when a first nucleotide does not occupy the nucleotide positiondefining the polymorphic site but not when the first nucleotide occupiesthe nucleotide position. Hybridization of the probe to thepolynucleotide sample is detected.

A diagnostic method of the invention may comprise (a) contacting apolynucleotide sample with one or more oligonucleotides that hybridizeunder stringent hybridization conditions to at least one polymorphism ofa gene of an N-glycan pathway or hexosamine pathway and detecting thehybridization; (b) detection of at least one, two, three, four, fivesix, seven, eight, nine, ten, fifteen, twenty, or twenty-fivepolymorphisms by amplification of the polynucleotide sample by, forexample, PCR; or (c) detection of at least two, three, four, five six,seven, eight, nine, ten, fifteen, twenty, or twenty-five polymorphism bydirect sequencing of the polynucleotide sample. In certain aspects, anindividual's risk for a disease disclosed herein (e.g. an autoimmunedisease) is diagnosed from the individual's genotype more particularlythe individual's N-glycan or hexosamine pathway genotype, in particularthe individual's GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5genotype. An individual who has at least one polymorphism statisticallyassociated with a disease disclosed herein (e.g. an autoimmune disease)possesses a factor contributing to either an increased or a decreasedrisk as compared to an individual without the polymorphism. Astatistical association of various polymorphisms (sequence variants)with an autoimmune disease is shown in the Examples. A genotype can bedetermined using any method capable of identifying nucleotide variation,e.g., nucleotide variation consisting of single nucleotide polymorphicsites. A number of suitable methods are described herein. For example,genotyping may be carried out using oligonucleotide probes specific tovariant GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 sequences. In anaspect, a region of the GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5genes which encompasses a polymorphic site of interest is amplifiedprior to, or concurrent with, the hybridization of probes complementaryto such sites. In the alternative, allele-specific amplification orextension reactions with allele-specific primers are used which supportprimer extension if the targeted allele is present. Typically, anallele-specific primer hybridizes to the gene such that the 3′ terminalnucleotide aligns with a polymorphic position.

In one aspect, the invention provides a method for detecting anindividual's increased or decreased risk for a disease disclosed herein(e.g. an autoimmune disease) by detecting the presence of one or moreSNPs in a polynucleotide sample of the individual, wherein the presenceof the SNP(s) indicates the individual's increased or decreased risk forthe disease. The SNPs can be any SNPs in a gene of the N-glycan pathwayor hexosamine pathway, including SNPs in exons, introns and/or upstreamand/or downstream regions, in particular SNPs in the promoter region.Examples of such SNPs include, but are not limited to, those discussedin detail herein and in the Examples. In one embodiment, the SNPspresent in a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5 locus (orlocus of another gene of the N-glycan and hexosamine pathway) areidentified by genotyping the GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/orMGAT5 SNPs. In certain embodiments, the genotype of one SNP can be usedto determine an individual's risk for an autoimmune disease. In otherembodiments, the genotypes of a plurality of SNPs can be used. In otherembodiments, certain combinations of SNPs at either the same ordifferent loci can be used.

The methods of the invention may also comprise detecting other markersand polymorphisms associated with disease disclosed herein in particularan autoimmune disease, more particularly MS or rheumatoid arthritis. Forexample, other markers and polymorphisms associated with MS include oneor more of the following: osteopontin polymorphisms, methionine synthasepolymorphisms; interferon receptor polymorphisms, myelin oligodendrocyteglycoprotein polymorphisms, PTPRC gene polymorphisms, early B-cellfactor gene (EBF-1) polymorphisms, APOE polymorphisms, CD24 genepolymorphisms, tumor necrosis factor β gene polymorphisms, interleukin(IL)-1B and IL-1 receptor antagonist (IL-1RN) gene polymorphisms;alpha-1 anti-trypsin; INF [alpha], [beta], [gamma]; TAP; LMP; and HLA-DPregion polymorphisms.

Evaluation of a candidate gene for association with various phenotypespertaining to an autoimmune disease is described in the Examples. Inaddition, design and execution of various types of association studieshave been described in the art; (see, e.g., Handbook of StatisticalGenetics, John Wiley and Sons Ltd.; Borecki and Suarez, 2001, Adv Genet42:45-66; Cardon and Bell, 200 1, Nat Rev Genet 2:91-99; and Risch,2000, Nature 405:847). Association studies have been used to evaluatecandidate genes for association with a phenotypic trait (e.g.,Thornsberry et al., 2001, Nature Genetics 28:286-289) and to performwhole genome scans to identify genes that contribute to phenotypicvariation.

Polymorphisms may be detected using analytical procedures well known toa person skilled in the art. Suitable methods for detection of allelicvariation are described in standard textbooks (e.g. “LaboratoryProtocols for Mutation Detection”, U. Landegren (ed), Oxford UniversityPress, 1996, and “PCR”, 2^(nd) Edition by Newton & Graham, BIOSScientific Publishers Limited, 1997, and reviewed by Nollau et al, Clin.Chem. 43, 114-1120, 1997). Generally, a method for detecting apolymorphism comprises a mutation discrimination technique, optionallyan amplification reaction, and a signal generation system.

Suitable mutation discrimination techniques include without limitationmutation detection techniques such as DNA sequencing, sequencing byhybridization, scanning (e.g. single-strand conformation polymorphismanalysis (SSCP), denaturing gradient gel electrophoresis (DGGE),temperature gradient gel electrophoresis (TGGE), cleavase, heteroduplexanalysis, chemical mismatch cleavage (CMC), enzymatic mismatchcleavage), solid phase hybridization [e.g. dot blots, multiple allelespecific diagnostic assay (MASDA)], reverse dot blots, oligonucleotidearrays (DNA Chips)], solution phase hybridization [eg. Taqman (U.S. Pat.Nos. 5,210,015 and 5,487,972 to Hoffman-LA Roche), and Molecular Beacons(Tyagi et al, 1996, Nature Biotechnology, 14: 303, WO 95/13399)],extension based techniques [e.g. amplification refractory mutationsystem linear extension (ALEX™) (EP Patent No. EP 332435), amplificationrefractory mutation system (ARMS™) (EP Patent NO. 332435; U.S. Pat. No.5,595,890; Newton et al Polynucleotides Research 17:2503, 1989);competitive oligonucleotide priming system (COPS) (Gibbs et al, 1989,Polynucleotide Research 17:2347); incorporation based techniques [e.g.mini-sequencing, arrayed primer extension (APEX)], restriction enzymebased techniques (e.g. restriction fragment length polymorphism,restriction site generating PCR), ligation based techniques(oligonucleotide ligation assay (OLA)—Nickerson et al, 1990, PNAS87:8923-8927); array-based tiling (EP 785280); Taqman allelicdiscrimination (Applied Biosystems), and other techniques known in theart.

Suitable signal generation or detection systems that may be used incombination with the mutation discrimination techniques include withoutlimitation fluorescence (fluorescence resonance energy transfer,fluorescence quenching, fluorescence polarization (UK Patent No.2228998), chemiluminescence, electrochemiluminescence, raman,radioactivity, colorimetric, hybridization protection assay, massspectrometry, and surface enhanced raman resonance spectroscopy (WO97/05280).

Various amplification methods known in the art can be used to detectnucleotide changes in a target polynucleotide. Suitable amplificationmethods include polymerase chain reaction (PCR), self sustainedreplication, branched DNA (b-DNA), ligase chain reaction (LCR),polynucleotide sequence based amplification (NASBA), and stranddisplacement amplification (SDA). Polymerase chain reaction (PCR), iswell known in the art [See U.S. Pat. Nos. 4,683,195; 4,683,202;4,965,188; PCR Applications, 1999, (Innis et al., eds., Academic Press,San Diego), PCR Strategies, 1995, (Innis et al., eds., Academic Press,San Diego); PCR Protocols, 1990, (Innis et al., eds., Academic Press,San Diego); and PCR Technology, 1989, (Erlich, ed., Stockton Press, NewYork); Abramson et al., 1993, Current Opinion in Biotechnology, 4:41-47;commercial vendors include PE Biosystems (Foster City, Calif.)].Reverse-transcription-polymerase chain reaction (RT-PCR) is also wellknown in the art and for example, is described in U.S. Pat. Nos.5,310,652; 5,322,770; 5,561,058; 5,641,864; and 5,693,517. Other knownamplification methods include the ligase chain reaction (Wu and Wallace,1988, Genomics 4:560-569); the strand displacement assay (Walker et al.,1992, Proc, Natl. Acad. Sci. USA 89:392-396, Walker et al. 1992,Polynucleotides Res. 20:1691-1696, and U.S. Pat. No. 5,455,166); andseveral transcription-based amplification systems, including the methodsdescribed in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491; thetranscription amplification system (TAS) (Kwoh et al., 1989, Proc. Natl.Acad. Sci. USA, 86:1173-1177); and self-sustained sequence replication(3 SR) (Guatelli et al., 1990, Proc. Natl. Acad. Sci, USA, 87:1874-1878and WO 92/08800). Methods that amplify a probe to detectable levels canalso be used, including QB-replicase amplification (Kramer et al., 1989,Nature, 339:401-402, and Lomeli et al., 1989, Clin. Chem.,35:1826-1831).

Certain methods of the invention may employ restriction fragment lengthanalysis, sequencing, hybridization, an oligonucleotide ligation assay,polymerase proofreading methods, allele-specific PCR and readingsequence data. Particular methods of the invention are described herein.

Genotyping can be carried out by detecting and analyzing mRNA underconditions when both maternal and paternal chromosomes are transcribed.Amplification of RNA can be carried out by first reverse-transcribingthe target RNA using, for example, a viral reverse transcriptase, andthen amplifying the resulting cDNA, or using a combined high-temperaturereverse-transcription-polymerase chain reaction (RT-PCR) [see forexample, U.S. Pat. Nos. 5,310,652; 5,322,770; 5,561,058; 5,641,864; and5,693,517 and Myers and Sigua, 1995, in PCR Strategies, supra, Chapter5).

Alleles can also be identified using allele-specific amplification orprimer extension methods which are based on the inhibitory effect of aterminal primer mismatch on the ability of a DNA polymerase to extendthe primer (see, for example, U.S. Pat. Nos. 5,137,806; 5,595,890;5,639,611; and U.S. Pat. No. 4,851,331). To detect an allele sequenceusing these methods, a primer complementary to the gene is selected suchthat the 3′ terminal nucleotide hybridizes at the polymorphic position.In the presence of the allele to be identified, the primer matches thetarget sequence at the 3′ terminus and the primer is extended. In theabsence of the allele, the primer has a 3′ mismatch relative to thetarget sequence and primer extension is either eliminated orsignificantly reduced. Using allele-specific amplification-basedgenotyping, identification of the alleles requires the detection of thepresence or absence of amplified target sequences. Methods for thedetection of amplified target sequences are well known in the art andinclude, for example, gel electrophoresis (see Sambrook et al., 1989,infra) and the probe hybridization assays described herein.

Allele-specific amplification-based methods of genotyping can facilitatethe identification of haplotypes. Essentially, the allele-specificamplification is used to amplify a region encompassing multiplepolymorphic sites from only one of the two alleles in a heterozygoussample. The SNP variants present within the amplified sequence are thenidentified by probe hybridization or sequencing.

A kinetic-PCR method in which the generation of amplified polynucleotideis detected by monitoring the increase in the total amount ofdouble-stranded DNA in the reaction mixture can also be used to identifypolymorphisms. The method is described, for example, in Higuchi et al.,1992, BioTechnology, 10:413-417; Higuchi et al., 1993, BioTechnology,11: 10261030; Higuchi and Watson, in PCR Applications, supra, Chapter16; U.S. Pat. Nos. 5,994,056 and 6,171,785; and European PatentPublication Nos. 487,218 and 512,334. The detection of double-strandedtarget DNA depends on the increased fluorescence that DNA-binding dyes,such as ethidium bromide or SYBR Green, exhibit when bound todouble-stranded DNA. An increase of double-stranded DNA produced fromthe synthesis of target sequences provides an increase in the amount ofdye bound to double-stranded DNA and a concomitant detectable increasein fluorescence. In the kinetic-PCR methods, amplification reactions arecarried out using a pair of primers specific for one of the alleles,such that each amplification indicates the presence of a particularallele. For example, by performing two amplifications, one using primersspecific for the wild-type allele and one using primers specific for themutant allele, the genotype of the sample with respect to that SNP canbe determined.

Alleles may also identified using probe-based methods, which rely on thedifference in stability of hybridization duplexes formed between a probeand its corresponding target sequence comprising an allele. Undersufficient stringent hybridization conditions, stable duplexes areformed only between a probe and its target allele sequence and not otherallele sequences. The presence of stable hybridization duplexes can bedetected by methods known in the art.

Probes suitable for use in the probe-based methods of the invention,which contain a hybridizing region either substantially complementary orexactly complementary to a polymorphic site of a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and/or MGAT5 gene or the complement thereof, or aSNP-containing polynucleotide of the invention, can be selected usingthe guidance provided herein and well known in the art. Similarly,suitable hybridization conditions (e.g., stringent conditions), whichdepend on the exact size and sequence of the probe, can be selectedempirically using the guidance provided herein and well known in the art(see, e.g., Polynucleotide Hybridization (B. D. Haines and S. F.Higgins. eds., 1984) and Sambrook et al., Molecular Cloning—A LaboratoryManual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 2000).

In aspects of probe-based methods for determining genotypes, multiplepolynucleotide sequences from the genes which comprise the polymorphicsites are amplified and hybridized to a set of probes under stringentconditions. The alleles present are determined from the pattern ofbinding of the probes to the amplified target sequences. In this aspect,amplification is carried out to provide sufficient polynucleotide foranalysis by probe hybridization. Therefore, primers are designed toamplify the regions of the genes encompassing the polymorphic sitesregardless of the allele present in the sample. Primers which hybridizeto conserved regions of the genes are used for allele-independentamplification. Suitable allele-independent primers can be selectedroutinely. Assays suitable for detecting hybrids formed between probesand target polynucleotide sequences in a sample are well known in theart, for example, immobilized target (dot-blot) and immobilized probe(reverse dot-blot or line-blot) assay formats. (See, for example,Schollen et al, Clin. Chem. 43: 18-23, 2997; Gilles et al, Nat.Biotechnol. 1999, 17(40:365-70; and U.S. Pat. Nos. 5,310,893; 5,451,512;5,468,613; and 5,604,099 for dot blot and reverse dot blot assayformats).

In an aspect of the invention, probe-based genotyping can be carried outusing a 5′-nuclease assay. (See for example, Holland et al., 1988, Proc.Natl. Acad. Sci. USA, 88:7276-7280, and U.S. Pat. Nos. 5,108,892,5,210,015, 5,487,972, and 5,804,375 describing 5′-nuclease assays). Inthis assay, labeled detection probes that hybridize within the amplifiedregion are added during the amplification reaction mixture. The probesare modified so that they do not act as primers for DNA synthesis. Theamplification is carried out using a DNA polymerase that possesses 5′ to3′ exonuclease activity. Any probe which hybridizes to the targetpolynucleotide downstream from the primer being extended in theamplification is degraded by the 5′ to 3′ exonuclease activity of theDNA polymerase. Therefore, the synthesis of a new target strand alsoresults in the degradation of a probe, and the accumulation of the probedegradation product provides a measure of the synthesis of targetsequences. Any method suitable for detecting the products may be used inthe assay. In an aspect of the invention, the probes are labeled withtwo fluorescent dyes, one of which is capable of quenching thefluorescence of the other dye. The dyes are attached to the probe (e.g.,one is attached to the 5′ terminus and the other is attached to aninternal site), such that quenching occurs when the probe is nothybridized and the cleavage of the probe by the 5′ to 3′ exonucleaseactivity occurs in between the two dyes. Amplification results incleavage of the probe between the dyes with the elimination of quenchingand an increase in the fluorescence which is observable from theinitially quenched dye. The accumulation of degradation product isdetermine by measuring the increase in reaction fluorescence. A5-nuclease assay may employ allele-specific amplification primers suchthat the probe is used only to detect the presence of amplified product.Alternatively, the 5′-nuclease assay can employ a target-specific probe.

The methods described above typically employ labeled oligonucleotides tofacilitate detection of the hybrid duplexes. Oligonucicotides can belabeled by incorporating a label detectable by spectroscopic,photochemical, biochemical, immunochemical, radiological, radiochemicalor chemical means. Useful labels and methods for labelingoligonucleotides are described herein.

Kit

The invention also provides a kit for carrying out a method of theinvention. Generally all of the features disclosed herein for themethods of the invention apply to the kits of the invention. A kit maytypically comprise two or more elements required for performing adiagnostic assay. Elements include but are not limited to compounds,reagents, containers, and/or equipment.

A kit can comprise reagents for assessing one or more SNP-containingpolynucleotides, variant polypeptides, N-glycans, and/or components ofan N-glycan pathway or a hexosamine pathway. In an aspect, the inventionprovides diagnostic tools, and kits for detecting, diagnosing, andpredicting a disease disclosed herein by monitoring SNP-containingpolynucleotides, variant polypeptides, N-glycans, or components of anN-glycan pathway or a hexosamine pathway.

The methods described herein may be performed by utilizing pre-packageddiagnostic kits comprising one or more specific binding agent (e.g.antibody) or polynucleotide which may be conveniently used, e.g., inclinical settings to screen and diagnose patients and to screen andidentify those individuals exhibiting a predisposition to developing adisease disclosed herein.

In an embodiment, a container is provided with a kit comprising abinding agent. By way of example, the kit may contain antibodies orantibody fragments or plant lectins (eg L-PHA, LEA) which bindspecifically to epitopes of a component of a N-glycan or hexosaminepathway or a polypeptide variant, and antibodies against the antibodiesor plant lectin labelled with a detectable substance. The kit may alsocontain microtiter plate wells, standards, assay diluent, wash buffer,adhesive plate covers, and/or instructions for carrying out a method ofthe invention using the kit.

In an aspect of the invention, the kit includes antibodies or fragmentsof antibodies which bind specifically to an epitope of one or moremarkers encoded by SNP-containing polynucleotides and means fordetecting binding of the antibodies to their epitope, either asconcentrates (including lyophilized compositions), which may be furtherdiluted prior to use or at the concentration of use, where the vials mayinclude one or more dosages.

In aspects of the invention, a kit may comprise a polynucleotide oroligonucleotide for detecting the presence of a polymorphism byhybridizing to the polymorphism under stringent conditions. In someembodiments, the oligonucleotide can be used as an extension primer ineither an amplification reaction such as PCR or a sequencing reaction,wherein a polymorphism is detected either by amplification orsequencing. In particular embodiments, the kit also comprisesamplification or sequencing primers which can, but need not, besequence-specific. The kit can also comprise reagents for labeling oneor more of the oligonucleotides, or comprise labeled oligonucleotides. Akit can optionally comprise reagents to detect the label.

In some embodiments, the kit can comprise one or more oligonucleotidesthat can be used to detect the presence of two or more predisposing orprotective disease associated polymorphisms or combinations ofpredisposing and protective polymorphisms or both.

One aspect of the invention provides kits for detecting the presence ofa first predisposing or protective disease associated polymorphism in agene in a polynucleotide sample of an individual whose susceptibility orrisk for a disease is being assessed. Thus, an aspect of the inventionprovides a kit including one or more oligonucleotides capable ofdetecting a polymorphism or SNP-containing polynucleotide, andinstructions for detecting the polymorphism or SNP-containingpolynucleotide, with the oligonucleotides and for correlating thedetection to the individual's risk for an autoimmune disease, packagedin one or more containers.

A kit may be designed to detect the level of polynucleotides encodingone or more polymorphism (e.g., SNP-containing polynucleotides).Accordingly, the invention relates to a kit for determining the presenceof a polymorphism or SNP-containing polynucleotide disclosed herein. Inan aspect, the invention provides a kit for the detection of apolymorphism comprising at a minimum, at least one polynucleotide of atleast 10 contiguous nucleotides of a gene of the N-glycan or hexosaminepathway co-localized in chromosomal regions associated with a diseasedisclosed herein, in particular a GCS1, GANAB, MAN1A1, MGAT1, MGAT2,and/or MGAT5 gene, or their complements, wherein at least onepolynucleotide contains at least one polymorphic sited associated withthe disease. In an embodiment of the invention, a kit comprisesoligonucleotides complementary to at least one SNP shown in FIGS. 11,22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47.

An aspect of the invention provides kits for detecting the presence of afirst predisposing or protective polymorphism in a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and MGAT5 gene, e.g., in a polynucleotide sample of anindividual whose risk for a disease disclosed herein (e.g, an autoimmunedisease, in particular MS or rheumatoid arthritis) is being assessed. Inan aspect, the invention provides a kit including one or moreoligonucleotides capable of detecting a polymorphism in the one or moregene of the N-glycan pathway or hexosamine pathway and instructions fordetecting the polymorphism with the oligonucleotides and for correlatingthe detection to the individual's risk for a disease disclosed herein(e.g, autoimmune disease, in particular MS or rheumatoid arthritis),packaged in one or more containers.

The invention relates to a kit useful for detecting the presence of apredisposing or a protective polymorphism in a gene of the N-glycanpathway or hexosamine pathway, in particular a GCS1, GANAB, MAN1A1,MGAT1, MGAT2, and/or MGAT5 gene, in a polynucleotide sample of anindividual whose risk or susceptibility for an autoimmune disease isbeing assessed. The kit can comprise one or more oligonucleotidescapable of detecting a predisposing or protective polymorphism in thelocus as well as instructions for using the kit to detect risk orsusceptibility to an autoimmune disease. In embodiments, theoligonucleotide or oligonucleotides each individually comprise asequence that hybridizes under stringent conditions to at least onepolymorphism. In some embodiments, the oligonucleotide oroligonucleotides each individually comprise a sequence that is fullycomplementary to a polynucleotide sequence comprising a polymorphismdescribed herein.

In one aspect, the kit can be used to detect the presence of apolymorphism by hybridization of a polynucleotide probe to thepolymorphism. Therefore, in an aspect of the invention, theoligonucleotides comprise at least one probe. In certain embodiments,the oligonucleotide hybridizes under stringent conditions to a region ofa gene comprising an an autoimmune disease-associated polymorphism. Inanother aspect, the polymorphism is a single nucleotide polymorphismcomprising a nucleotide at a particular nucleotide position. Understringent conditions, the oligonucleotide hybridizes to a region of agene comprising the single nucleotide polymorphism with a signal tonoise ratio that is at least 2 times (e.g., at least 5 times or at least10 times) the signal to noise ratio at which the oligonucleotidehybridizes to the region of the gene in the absence of the polymorphismat the nucleotide position. The oligonucleotide is typically fullycomplementary to the region of the gene comprising the polymorphism, andtypically comprises at least about 10 to 30 contiguous nucleotidescomplementary to the gene.

To facilitate detection of a polymorphism the oligonucleotides in a kitoptionally comprise a label (e.g., an isotopic, fluorescent,fluorogenic, luminescent or colorimetric label). In some aspects, thelabel itself directly produces a detectable signal (e.g., a fluorescentlabel). In other aspects, the kit also includes a reagent that detectsthe label (e.g., an enzyme that cleaves a colorimetric label and thelike).

In an aspect of a kit of the invention, the oligonucleotides compriseprimers. The primer(s) can be used to detect a polymorphism of theinvention in an allele-specific amplification or extension reaction. Theprimer(s) can be used to amplify a region of a gene comprising thepolymorphism for subsequent detection of the polymorphism byhybridization, sequencing, or the like. Thus, in one aspect, theoligonucleotides comprise amplification primers, wherein theamplification primers amplify a polynucleotide sequence comprising thepolymorphism. The oligonucleotides can also comprise primers that flankthe polymorphism.

A kit can optionally be used to detect more than one polymorphism(simultaneously or sequentially). Thus, in aspects of the invention, thekit also includes one or more second oligonucleotides capable ofdetecting a second polymorphism (and optionally third, fourth, fifth,etc. oligonucleotides capable of detecting third, fourth, fifth, etc.polymorphisms). A second polymorphism can be at the same polymorphicsite as a first polymorphism, or at a different polymorphic site, andcan be protective or predisposing.

The oligonucleotides in a kit can be optionally immobilized on asubstrate. The substrate can be, for example, a planar substrate or abeaded substrate.

In an aspect, a kit comprises in a package a restriction enzyme capableof distinguishing alternate nucleotides at the polymorphism site or alabeled oligonucleotide being sufficiently complementary to thepolymorphism site and capable of distinguishing the alternatenucleotides at the polymorphism site (i.e. probes). A kit may alsocomprise primers to amplify a region surrounding the polymorphism site,a polymerization agent and instructions for using the kit to determinegenotype.

The invention also relates to a kit comprising a container unit andcomponents for practicing a method of the invention. A kit can containoligonucleotide probes specific for alleles as well as instructions fortheir use to determine risk or susceptibility for an autoimmune disease.In some cases, a kit may comprise detection probes fixed to anappropriate support membrane. The kit can also contain amplificationprimers for amplifying regions of a locus encompassing the polymorphicsites, as such primers are useful in aspects of the invention.Alternatively, useful kits can contain a set of primers comprising anallele-specific primer for the specific amplification of alleles. Otheroptional components of the kits include additional reagents used in thegenotyping methods as described herein. For example, a kit additionallycan contain an agent to catalyze the synthesis of primer extensionproducts, substrate nucleoside triphosphates, reagents for labelingand/or detecting polynucleotides (for example, an avidin-enzymeconjugate and enzyme substrate and a chromogen if the label is biotin)and appropriate buffers for amplification or hybridization reactions.

Array

The invention relates to an array, a support with immobilizedoligonucleotides, useful for practicing the present method. A usefularray can contain oligonucleotide probes specific for alleles or certaincombinations of alleles described herein or for SNP-containingpolymorphisms. The oligonucleotides can be immobilized on a substrate.The oligonucleotides may be labeled. In some embodiments, the array canbe a micro-array. In some embodiments, the array can comprise one ormore oligonucleotides used to detect the presence of two or more allelesor certain combinations of alleles. Oligonucleotide(s) can be arrangedin an array of other oligonucleotides which can be used to detect otherpolymorphisms.

In aspects of the invention, arrays are provided for detecting thepresence of one or more predisposing and/or protectivedisease-associated polymorphisms in a gene (e.g, autoimmune diseaseassociated polymorphisms), for example, in a polynucleotide sample of anindividual whose risk for an autoimmune disease is being assessed. In aparticular aspect, the array comprises a substrate and a plurality ofoligonucleotides, each oligonucleotide capable of hybridizing to aregion of the gene comprising at least one polymorphism. Thehybridization detects the presence of the polymorphism, and provides anindication of the individual's risk for a disease (e.g., autoimmunedisease). Typically, the array is used for detecting the presence of aplurality of polymorphisms, e.g., multiple alleles at a singlepolymorphic site and/or different polymorphic sites.

Generally all of the features noted for the method and kit aspects ofthe invention apply to an array of the invention. For example, the oneor more polymorphisms preferably comprise one or more single nucleotidepolymorphisms. For example, the polymorphism may be one or more SNPshown in FIGS. 11, 22, and 23, and SEQ ID NOs.:5, 6, 7, 8, 9, and 35-47.

The invention in particular contemplates an array that can be used todetect the presence of one or more SNPs comprising oligonucleotideswhich are capable of hybridizing under stringent conditions to a regionof a gene comprising a single nucleotide polymorphism with a signal tonoise ratio that is at least 2, 5 or 10 times that at which theoligonucleotide hybridizes to a region of the gene comprising anothersingle nucleotide polymorphisms. Typically, one oligonucleotide is usedto detect one SNP; that is, each oligonucleotide is capable ofhybridizing to a distinct SNP.

A plurality of oligonucleotides may be immobilized on a substrate, e.g.,a planar substrate, a membrane, a glass slide, or the like. Typically,each of the plurality of oligonucleotides is immobilized at a known,predetermined position on the substrate. Each oligonucleotide may bebound (e.g., electrostatically or covalently bound, directly or via alinker) to the substrate at a unique location. In order to facilitatedetection of polymorphisms by specific hybridization with theoligonucleotides, each of the oligonucleotides is typically fullycomplementary to a region of a gene comprising one of the polymorphisms,and each of the plurality of oligonucleotides comprises at least about10 to 30 contiguous nucleotides complementary to the gene. Anoligonucleotide may optionally comprise a label that facilitatesdetection of hybridization between the oligonucleotide and thepolymorphism.

An array can be part of a system. Thus, the invention provides a systemcomprising an array of the invention and system instructions thatcorrelate the detection of the presence of one or more predisposing orprotective disease-associated polymorphisms (e.g., autoimmune-associatedpolymorphisms) to the individual's risk for the disease.

Methods of making, using, and analyzing arrays such as micro-arrays arewell known in the art (see, e.g., Wang et al., 1998, Science280:1077-82; Lockhart and Winzeler, 2000, Nature 405:827-836; and Scherfet al., 2000, Nat Genet. 24:236). Arrays can be formed (e.g., printed),using commercially available instruments (e.g., a GMS 417 Arrayer,Affymetrix, Santa Clara, Calif.). Suitable solid supports arecommercially available and include without limitation membranes (e.g.,nylon, PVDF, and nitrocellulose membranes) and surface-modified andpre-coated slides with a variety of surface chemistries (e.g., fromTeleChem International (www.arrayit.com), Corning, Inc. (Corning, N.Y.),or Greiner Bio-One, Inc. (www.greinerbiooneinc.com). Further, customarrays of polynucleotides are commercially available (e.g., from AgilentTechnologies (CA, USA) and from TeleChem International (CA,USA)(www.arrayit.com)).

Automated Methods

Various automated systems can be used to perform some or all of themethods of the invention. In addition, digital or analog systems, forexample, comprising a digital or analog computer, can also control avariety of other functions such as a user viewable display to permitviewing of method results by a user and/or control of output features.For example, particular methods described herein may be implemented on acomputer program or programs. The programs may correlate detection ofthe presence of one or more predisposing or protective polymorphisms toan individual's risk for a disease disclosed herein, in particular anautoimmune disease, more particularly MS. Therefore, the inventioncontemplates digital systems, including computers, computer readablemedia, and/or integrated systems comprising instructions (e.g., embodiedin appropriate software) for performing the methods of the invention. Inan aspect, the invention provides a digital system comprisinginstructions for correlating detection of the presence of one or morepredisposing or protective polymorphisms to an individual's risk for adisease disclosed herein in particular an autoimmune disease, moreparticularly MS. A digital system may also include informationcorresponding to individual genotypes for a set of genetic markers,phenotypic information, and the like. The system may also assist in thedetection of polymorphisms by, for example, controlling a microarrayscanner.

Standard desktop applications can be adapted to the present invention byinputting data which is loaded into the memory of a digital system, andperforming an operation on the data. Such applications include wordprocessing software such as Microsoft Word, database software such asMicrosoft Excel, and/or database programs such as Microsoft Access. Forexample, systems including these software applications containingappropriate genotypic information, associations between phenotype andgenotype, and other relevant information may be used in conjunction witha user interface (e.g., a GUI in a standard operating system such as aWindows, Macintosh or LINUX system) to perform any analysis notedherein, or simply to retrieve data (e.g., in a spreadsheet) to be usedin the methods disclosed herein. A system may include a digital computerwith software for performing association analysis and/or riskprediction, and also data sets entered into the software systemcomprising genotypes for a set of genetic markers, phenotypic values andthe like. The systems of the invention can use commercially availablecomputers.

The methods of the invention may also be embodied within the circuitryof an application specific integrated circuit (ASIC) or programmablelogic device (PLD). In such applications, a method of the invention isembodied in a computer readable descriptor language that can be used tocreate an ASIC or PLD. The methods of the invention can also be embodiedwithin the circuitry or logic processors of other digital apparatus,such as PDAs, laptop computer systems, displays, image editingequipment, and the like.

Therapeutic Applications

Combinations of genetic defects and environmental factors that lead torelatively small reductions in Mgat5 expression represent candidatesusceptibility factors for MS and other autoimmune diseases. Mgat5glycan expression is dependent on the activity of multiple genes in twobiochemical pathways: the N-glycan pathway and the hexosamine pathway.Genetic hypomorphism in the hexosamine and N-glycan pathways in mammalsmay increase susceptibility to autoimmune diseases by limiting Mgat5glycan expression in T cells. Genes in these two pathways aredisproportionately found in chromosomal regions previously associatedwith autoimmune diseases such as MS. White blood cells from patientsdisplay structural abnormalities in N-glycan profiles. DNA sequencingidentified multiple SNPs in 5-glycosylation genes associated withautoimmune diseases such as MS. Observed genetic and structural N-glycanprocessing defects are associated with attenuated Mgat5 glycanup-regulation during T cell proliferation. Genetically determineddefects in Mgat5 glycan expression are common in patients withautoimmune diseases (e.g., the defects were observed in greater than 85%of tested MS patients). In the animal models described herein, alteredMgat5 modified glycan expression leads to T cell hyper-proliferation,preferential Th1 differentiation and spontaneous autoimmune CNSdemyelinating disease, indicating that the genotypic and phenotypicdefects in Mgat5 modified glycan regulation identified in autoimmunedisease patients (e.g, MS patients) directly promotes disease.

Mgat5-modified N-glycans are sensitive to changes in the intracellularconcentrations of UDP-GlcNAc, the sugar donor synthesized de novo fromglucose via the hexosamine pathway. Metabolic supplementation of thehexosamine pathway in T cells raises Mgat5 modified glycan levels;inhibits proliferation, Th1 differentiation and EAE; and can reverseMgat5 modified glycan down regulation induced by blockade of proximalN-glycan processing. Vitamin D3 up-regulates MGAT5 mRNA expression inhepatoma cells, inhibits T cell activation, T_(H)1 differentiation, EAEand is an environmental factor whose deficiency is associated with MS.Addition of Vitamin D3 to Jurkat T cells or human PBMCs enhanced Mgat5modified glycan expression, synergized with anti-CD3 in up-regulatingmgat5 modified glycan levels, rescued swainsonine induceddown-regulation of Mgat5 modified glycans and reversed attenuated Mgat5N-glycan expression in MS patients.

Chemical blockade of Mgat5 glycan expression reversed Vitamin D3 inducedinhibition of T cell proliferation, indicating that Vitamin D3negatively regulates T cell function by enhancing Mgat5 modified glycanexpression. Thus, Vitamin D3 levels and metabolic flux through thehexosamine pathway provide two independent mechanisms for environmentaland therapeutic modulation of Mgat5 modified glycan expression anddisease promotion by the identified SNPs. The data herein define geneticand environmental regulation of Mgat5 glycan expression as a majorsusceptibility factor for autoimmune diseases such as MS.

Treatments, Compositions, and Kits Comprising Agonists, Regulators,Metabolites Etc.

The invention provides a method of treating a disease disclosed herein,in particular an autoimmune disease, more particularly MS, in a subjectcomprising modulating one or more of N-glycans (e.g., expression orlevels), N-glycan processing, an N-glycan pathway, and a hexosaminepathway. In an aspect, N-glycans, N-glycan processing, an N-glycanpathway, and/or a hexosamine pathway are modulated by modulating one ormore of glucosidase I (GI), mannosidase I (MI), mannosidase II(MII/MIIx), MGAT1, MGAT2, and MGAT5. In a particular embodiment,N-glycans, N-glycan processing, an N-glycan pathway, and/or a hexosaminepathway are modulated by administering a substance that raises N-glycanlevels or up-regulates MGAT5 expression. A substance may be an agonistto polypeptides encoded by genes of the N-glycan pathway or hexosaminepathway associated with a disease disclosed herein, a compound thatchange the concentration of upstream regulators or downstream effectormolecules of a polypeptide encoded by a gene of the N-glycan pathway orhexosamine pathway.

Agonists to polypeptides encoded by genes of the N-glycan pathway orhexosamine pathway associated with a disease disclosed herein can beantibodies, peptides, proteins, polynucleotides, small organicmolecules, or polymers. Agonists may be prepared as a composition with apharmaceutically acceptable carrier, vehicle or diluent. Antibodies canbe prepared by conventional methods and peptides, proteins,polynucleotides, small organic molecules, and polymers may be identifiedby combinatorial methods. Agonists may be prepared as a composition witha pharmaceutically acceptable carrier, vehicle or diluent.

An aspect of the invention is directed towards a method to treat adisease disclosed herein, in particular an autoimmune disease, moreparticularly MS. The method may comprise selecting a subject diagnosedwith an autoimmune disease and administering to the subject an agonist,in particular an agonist of the expression or activity of GCS1, GANAB,MAN1A1, Mgat1, Mgat2, and/or Mgat5. The agonist can be administered at aconcentration suitable to reduce the effects of the disease. Theconcentration of the agonist may be less than about 100 mM, about 10 mM,about 1 mM, 100 μM, about 10 μM, about 1 μM, about 0.1 μM, about 0.01μM, about 0.001 μM or about 0.0001 μM.

Another aspect embodiment of the invention is directed to the use ofcompounds that change the concentration of upstream regulators ordownstream effector molecules of a polypeptide encoded by a gene of theN-glycan pathway or hexosamine pathway, in treating or preventing adisease disclosed herein, in particular an autoimmune disease, moreparticularly MS. The method can comprise selecting a subject diagnosedwith the disease, and administering to the mammal one or more agonist,in particular a Mgat5 agonist. The concentration of agonist can be lessthan about 100 μM, about 10 μM, about 1 μM, about 0.1 μM, about 0.01 μM,about 0.001 μM or about 0.0001 μM.

In an embodiment of the invention, a method of treatment comprisesincreasing N-glycan levels, in particular Mgat5 modified glycan levels,by administering an agonist of Mgat5 and/or metabolites to raiseUDP-GlcNAc levels in the cell.

The invention also provides methods and compositions for treating orpreventing a disease discussed herein in a subject comprising increasingin the subject expression or amount of one or more of (a) N-glycans, inparticular, Mgat5 modified glycans and/or polylactosamine modifiedglycans, and (b) a component of the N-glycan pathway or hexosaminepathway. Aspects of the methods of the invention comprise administeringto a subject one or more of the following: N-glycans (e.g., Mgat5modified glycans or polylactosamine modified glycans); an agonist of acomponent of the N-glycan or hexosamine pathways (e.g., an agonist of anenzyme of the N-glycan pathway, especially Mgat5); a substance thatincreases expression or synthesis of a component of the N-glycan orhexosamine pathways (e.g., an enzyme of the N-glycan pathway, especiallyMgat5); an acceptor for an enzyme of the N-glycan pathway (e.g., anacceptor for Mgat5); a donor for an enzyme of the N-glycan pathway(e.g., a donor for Mgat5), or a hexosamine pathway metabolites.

In an aspect, the invention provides methods and compositions fortreating or preventing a disease discussed herein, in particular anautoimmune disease, more particularly an autoimmune demyelinatingdisease, most particularly multiple sclerosis, in a subject comprisingincreasing in the subject expression or amount of Mgat5, Mgat5 modifiedglycans and/or polylactosamine modified glycans or a polypeptide or agene or polypeptide comprising a polymorphism identified using a methodof the invention. The expression or amount of Mgat5 modified glycans orpolylactosamine modified glycans can be increased by administering Mgat5modified glycans or polylactosamine modified glycans to the subject oran agonist of Mgat5, or increasing expression or synthesis of Mgat5, anacceptor for Mgat5, or a donor for Mgat5.

In an aspect of a preventive and therapeutic method of the invention,expression of Mgat5 modified glycans are increased by administering anagonist of Mgat5. In a particular aspect, the agonist up-regulates MGAT5polynucleotides, in particular MGAT5 mRNA. Examples of agonists of MGAT5expression include insoluble lipid vitamins agonists such as Vitamin D3,retinoic acid, analogs and derivatives thereof, and the like. Inembodiments of the methods and compositions of the invention a VitaminD3 compound including vitamin D3, 1-hydroxy Vitamin D3, and 1,25dihydroxy-Vitamin D3 are utilized.

In another aspect of a preventive and therapeutic method of theinvention, the amount of a sugar donor for Mgat5 is increased. A sugardonor concentration can be increased by administering one or more of asugar donor, a metabolite of pathways for synthesis of the sugar donoror precursors thereof, regulators or agonists of a sugar donor orpathway for synthesis of a sugar donor, or hexosamine pathwaymetabolites. A sugar donor may be a nucleotide sugar,dolichol-phosphate-sugar or dolichol-pyrophosphate-oligosaccharide, forexample, cytidine or uridine diphospho-N-acetylglucosamine (CDP-GlcNAcor UDP-GlcNAc), or derivatives or analogs thereof. Metabolites for usein the methods of the invention include without limitation one or moreof nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP), nucleosides (e.g.cytidine or uridine), nucleobases (e.g., uracil, cytosine) sugars (e.g.glucose), acetoacetate, glutamine, glucosamine, N-acetyl-glucosamine(GlcNAc), and analogs and derivatives thereof.

In particular aspects of the invention a sugar donor comprises one ormore metabolite of a pathway for synthesis of a sugar donor, wherein themetabolite is selected from the group consisting of nucleotides (e.g,UMP, UDP, UTP, CMP, CDP, or CTP); nucleosides (e.g. cytidine oruridine), nucleobases (e.g., uracil, cytosine), sugars (e.g. glucose),acetoacetate, glutamine, glucosamine or GlcNAc or analogs andderivatives thereof.

In some embodiments the metabolites comprise a nucleotide, nucleoside,nucleobase, a sugar, and/or a combination of a nucleotide, nucleoside,nucleobase, and a sugar, more particularly uridine or cytidine and asugar, most particularly UDP, uracil, uridine, GlcNAc, and/or analogs orderivatives thereof.

In certain embodiments an analog or derivative of a metabolite is used.For example, an acetylated GlcNAc, peracetylated GlcNAc, or GlcNActetraacetate (e.g., N-acetyl-beta-D-glucosamine tetraacetate oralpha-D-N-acetylglucosamine tetraacetate) may be used in any of themethods of the invention.

In an embodiment of the invention, a method is provided for treating orpreventing a disease discussed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease, most particularly multiple sclerosis, in asubject comprising administering a therapeutically effective amount ofone or more hexosamine pathway metabolites.

In an embodiment of the invention, a method is provided for treating orpreventing a disease discussed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease, most particularly multiple sclerosis, in asubject comprising administering a therapeutically effective amount ofglucosamine, GlcNAc, and/or vitamin D3 compounds, or analogs orderivatives thereof.

In a particular embodiment of the invention, a method is provided fortreating or preventing a disease discussed herein, in particular anautoimmune disease, more particularly rheumatoid arthritis or anautoimmune demyelinating disease, most particularly multiple sclerosis,comprising administering a therapeutically effective amount of one ormore of GlcNAc or an analog or derivative thereof, a Vitamin D3compound, a nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP),nucleoside (e.g. cytidine or uridine), a nucleobase (e.g., uracil,cytosine), sugar (e.g., glucose), acetoacetate, glutamine, andglucosamine.

In another particular embodiment of the invention, a method is providedfor treating or preventing a disease discussed herein, in particular anautoimmune disease, more particularly rheumatoid arthritis or anautoimmune demyelinating disease, most particularly multiple sclerosis,comprising administering a therapeutically effective amount of one ormore of GlcNAc or an analog or derivative thereof, a Vitamin D3compound, nucleoside (e.g. cytidine or uridine), a nucleobase (e.g.,uracil, cytosine), sugar (e.g., glucose), acetoacetate, glutamine, andglucosamine.

In a further particular embodiment of the invention, a method isprovided for treating or preventing a disease discussed herein, inparticular an autoimmune disease, more particularly rheumatoid arthritisor an autoimmune demyelinating disease, most particularly multiplesclerosis, comprising administering a therapeutically effective amountof one or more of acetylated GlcNAc, uracil, uridine, and a Vitamin D3compound.

In an aspect of the invention, a method is provided for treating orpreventing a disease discussed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease, most particularly multiple sclerosis, comprisingadministering synergistically therapeutically effective amounts of oneor more of GlcNAc, or an analog or derivative thereof, a Vitamin D3compound, a nucleotide (e.g, UMP, UDP, UTP, CMP, COP, or CTP),nucleoside (e.g. cytidine or uridine), nucleobase (e.g., uracil,cytosine), sugar (e.g., glucose), acetoacetate, glutamine, andglucosamine.

In an embodiment of the invention, a method is provided for treating orpreventing a disease discussed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease, most particularly multiple sclerosis, comprisingadministering synergistically therapeutically effective amounts of oneor more of GlcNAc, or an analog or derivative thereof, a Vitamin D3compound, a nucleoside (e.g. cytidine or uridine) and glucosamine, inparticular GlcNAc, uridine, and Vitamin D3.

The invention also contemplates a method of increasing Mgat5 modifiedglycans in a cell comprising administering to the cell an amount of oneor more of GlcNAc or an analog or derivative thereof, a Vitamin D3compound, nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside(e.g. cytidine or uridine), nucleobase (e.g., uracil, cytosine), sugar(e.g., glucose), acetoacetate, glutamine, and/or glucosamine, to providean increase in the MGAT5 modified glycans which is greater compared toadministration of GlcNAc, Vitamin D3 compound, a nucleotide (e.g, UMP,UDP, UTP, CMP, CDP, or CTP), a nucleoside (e.g. cytidine or uridine), ora nucleobase (e.g., uracil, cytosine) sugar (e.g., glucose),acetoacetate, glutamine, and/or glucosamine, alone.

The invention further provides a combination therapy comprisingadministering GlcNAc and/or glucosamine or analogs or derivativesthereof, and one or more of a Vitamin D3 compound a nucleotide (e.g,UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g. cytidine or uridine),nucleobase (e.g., uracil, cytosine), sugar (e.g., glucose),acetoacetate, or glutamine, for the treatment and/or prevention of anautoimmune disease in particular rheumatoid arthritis or a autoimmunedemyelinating disease, in particular multiple sclerosis.

Another aspect of this invention is a method of treating a diseasediscussed herein, in particular an autoimmune disease, more particularlyrheumatoid arthritis or an autoimmune demyelinating disease, mostparticularly multiple sclerosis, comprising the steps of administeringto a patient, either together or separately, GlcNAc and/or glucosamineanalogs or derivatives thereof, and one or more of a nucleotide (e.g,UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g. cytidine or uridine),nucleobase (e.g., uracil, cytosine), sugar (e.g., glucose),acetoacetate, or glutamine.

An additional aspect of the invention is directed to methods for theprevention of a disease disclosed herein, in particular an autoimmunedisease, more particularly MS or rheumatoid arthritis. The methods maycomprise selecting a subject, and administering to the mammal anagonist, in particular a Mgat5 agonist. The agonist is preferablyadministered at a concentration suitable to reduce the occurrence oreffects of the disease relative to a subject which did not receive theadministration. The concentration of the agonist is preferably less thanabout 100 mM, about 10 mM, about 1 mM, 100 μM, about 10 μM, about 1 μM,about 0.1 μM, about 0.01 μM, about 0.001 μM or about 0.0001 μM.

In the methods of the invention the administering step can be performedby any acceptable means, including oral, inhalation, topical,intravenous, intraperitoneal, and intramuscular administration.

In an embodiment, this invention provides methods of treating orpreventing a disease discussed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or an autoimmunedemyelinating disease, most particularly multiple sclerosis, in a mammalcomprising the step of administering to the mammal any of thecompositions and combinations described herein.

The invention includes combination treatments providing synergisticactivity or delivering synergistically effective amounts of one or morehexosamine pathway metabolite; GlcNAc or glucosamine or analogs orderivatives thereof and other hexosamine pathway metabolites; an agonistof a component of the N-glycan pathway or hexosamine pathway; and/or,one or more of Vitamin D3, GlcNAc or an analog or derivative thereof, ofa nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g.cytidine or uridine), nucleobase (e.g., uracil, cytosine), sugar (e.g.,glucose), acetoacetate, glutamine, or glucosamine. Compositions suitablefor use in the present invention include compositions wherein the activeingredients are contained in a synergistically effective amount. Such acomposition comprises sufficient amounts of each component to achieve adesired result that is greater than the result achieved with eachcomponent on its own.

An aspect of this invention is the use of a sugar donor, metabolite,regulator or agonist discussed herein, in particular UDP-GlcNAc, or acombination of GlcNAc and/or glucosamine or analogs or derivativesthereof, and one or more hexosamine pathway metabolite, or GlcNAc and/orglucosamine or analogs or derivatives thereof and one or more of VitaminD3, a nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside(e.g. cytidine or uridine), nucleobase (e.g., uracil, cytosine), sugar(e.g., glucose), acetoacetate, or glutamine, in the preparation of amedicament, to treat or prevent a disease discussed herein, inparticular an autoimmune disease, more particularly rheumatoid arthritisor an autoimmune demyelinating disease, most particularly multiplesclerosis.

The present invention provides a combination therapy pharmaceuticalcomposition comprising GlcNAc or an analog or derivative thereof, andone or more hexosamine pathway metabolite, or GlcNAc and/or glucosamineor analogs or derivatives thereof, and one or more of a Vitamin D3compound, a nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP),nucleoside (e.g. cytidine or uridine), nucleobase (e.g., uracil,cytosine), sugar (e.g., glucose), acetoacetate, or glutamine.Accordingly, the invention relates to a multi-component compositioncomprising GlcNAc or an analog or derivative thereof, and one or morehexosamine pathway metabolite, or GlcNAc and/or glucosamine or analogsor derivatives thereof, and one or more of a Vitamin D3 compound, anucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g.cytidine or uridine), nucleobase (e.g., uracil, cytosine), sugar (e.g.,glucose), acetoacetate, or glutamine, and a pharmaceutically acceptablecarrier, diluent, or excipient. In an embodiment, the multi-componentcomposition comprises additive amounts, in particular synergisticamounts, of GlcNAc or an analog or derivative thereof, and one or morehexosamine pathway metabolite, or GlcNAc and/or glucosamine or analogsor derivatives thereof, and one or more of a Vitamin D3 compound, anucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g.cytidine or uridine), nucleobase (e.g., uracil, cytosine), sugar (e.g.,glucose), acetoacetate, or glutamine.

In accordance with another aspect, a composition is provided comprisinga combination of GlcNAc or an analog or derivative thereof, and one ormore hexosamine pathway metabolite, or GlcNAc and/or glucosamine oranalogs or derivatives thereof, and one or more of a Vitamin D3compound, a nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, or CTP),nucleoside (e.g. cytidine or uridine), nucleobase (e.g., uracil,cytosine), sugar (e.g., glucose), acetoacetate, or glutamine effectiveto exert a synergistic effect in preventing and/or treating rheumatoidarthritis or a CNS demyelinating autoimmune disease, in particularmultiple sclerosis. In an embodiment, the composition comprises a GlcNAcor an analog or derivative thereof, and one or more hexosamine pathwaymetabolite, or GlcNAc and/or glucosamine or analogs or derivativesthereof, and one or more of a Vitamin D3 compound, a nucleotide (e.g,UMP, UDP, UTP, CMP, CDP, or CTP), nucleoside (e.g. cytidine or uridine),nucleobase (e.g., uracil, cytosine), sugar (e.g., glucose),acetoacetate, or glutamine, in doses that are at least 1 to 10 fold, 2to 10 fold, or 5 to 10 fold lower than the doses of each componentrequired to prevent and/or treat the disease.

The compositions of the invention preferably contain a pharmaceuticallyacceptable carrier diluent, or excipient suitable for rendering thecompounds administrable orally, intranasally, parenterally,intravenously, intradermally, intramuscularly or subcutaneously,rectally, via inhalation or via buccal administration, or transdermally.The active ingredients may be admixed or compounded with anyconventional pharmaceutically acceptable carrier or excipient. It willbe understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agents may be utilized for preparingand administering the pharmaceutical compositions of the presentinvention. Illustrative of such methods, vehicles and carriers are thosedescribed, for example, in Remington: The Science and Practice ofPharmacy, 21^(st) Edition. University of the Sciences in Philadelphia(Editor), Mack Publishing Company. Those skilled in the art, having beenexposed to the principles of the invention, will experience nodifficulty in determining suitable and appropriate vehicles, excipientsand carriers or in compounding the active ingredients therewith to formthe pharmaceutical compositions of the invention.

The present invention provides a process for making a pharmaceuticalcomposition comprising combining a GlcNAc or an analog or derivativethereof, and one or more hexosamine pathway metabolite, or GlcNAc and/orglucosamine or analogs or derivatives thereof, and one or more of aVitamin D3 compound, a nucleotide (e.g, UMP, UDP, UTP, CMP, CDP, orCTP), nucleoside (e.g. cytidine or uridine), nucleobase (e.g., uracil,cytosine), sugar (e.g., glucose), acetoacetate, or glutamine, and apharmaceutically acceptable carrier or excipient.

The present invention provides a dietary supplement compositioncomprising one or more of a Vitamin D3 compound, retinoic acid,hexosamine pathway metabolite, nucleotides (e.g. UMP, UDP, UTP, CMP,CDP, or CTP), nucleosides (e.g. cytidine or uridine), nucleobases (e.g.,uracil, cytosine) sugars (e.g. glucose), acetoacetate, glutamine,glucosamine, N-acetyl-glucosamine (GlcNAc), or nutraceuticallyacceptable derivatives thereof. The invention also provides a method ofmanufacturing a nutritional or dietary supplement composition of theinvention effective in the prevention, stabilization, reversal and/ortreatment of a disease disclosed herein comprising combining one or moreof a Vitamin D3 compound, retinoic acid, hexosamine pathway metabolite,nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP), nucleosides (e.g.cytidine or uridine), nucleobases (e.g., uracil, cytosine) sugars (e.g.glucose), acetoacetate, glutamine, glucosamine, N-acetyl-glucosamine(GlcNAc), or nutraceutically acceptable derivatives thereof with anutraceutically acceptable carrier.

In an aspect, the invention provides a dietary supplement for mammalianconsumption, particularly human consumption for the purpose of treatingor preventing a disease disclosed herein, in particular an autoimmunedisease, more particularly rheumatoid arthritis or MS, comprising one ormore of Vitamin D, retinoic acid, hexosamine pathway metabolites,nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP), nucleosides (e.g.cytidine or uridine), nucleobases (e.g., uracil, cytosine) sugars (e.g.glucose), acetoacetate, glutamine, glucosamine, N-acetyl-glucosamine(GlcNAc), or nutraceutically acceptable derivatives thereof.

In another aspect, the invention provides a dietary supplement formammalian consumption, particularly human consumption for the purpose ofimproving N-glycan regulation or function, modulating the immuneresponse (e.g. by modulating T cell function or cytokine production),reducing inflammation, and/or improving CNS function in a diseasedisclosed herein, in particular an autoimmune disease, more particularlyMS, comprising one or more of a Vitamin D3 compound, retinoic acid,hexosamine pathway metabolites, nucleotides (e.g. UMP, UDP, UTP, CMP,CDP, or CTP), nucleosides (e.g. cytidine or uridine), nucleobases (e.g.,uracil, cytosine) sugars (e.g. glucose), acetoacetate, glutamine,glucosamine, N-acetyl-glucosamine (GlcNAc), or nutraceuticallyacceptable derivatives thereof.

In another aspect, the invention provides a supplement comprising one ormore of a Vitamin D3 compound, retinoic acid, hexosamine pathwaymetabolites, nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP),nucleosides (e.g. cytidine or uridine), nucleobases (e.g., uracil,cytosine) sugars (e.g. glucose), acetoacetate, glutamine, glucosamine,N-acetyl-glucosamine (GlcNAc), or nutraceutically acceptablederivatives, thereof for improving N-glycan regulation or function,modulating the immune response (e.g. by modulating T cell function orcytokine production), reducing inflammation, and/or improving CNSfunction of individuals who suffer from a disease disclosed herein andwho have taken the supplement.

A dietary supplement of the invention is preferably pleasant tasting,effectively absorbed into the body and provides substantial therapeuticeffects.

This invention also includes a regimen for supplementing a healthyindividual's diet to prevent a disease disclosed herein, in particularan autoimmune disease, more particularly MS or rheumatoid arthritis, byadministering one or more of a Vitamin D3 compound, retinoic acid,hexosamine pathway metabolites, nucleotides (e.g. UMP, UDP, UTP, CMP,CDP, or CTP), nucleosides (e.g. cytidine or uridine), nucleobases (e.g.,uracil, cytosine) sugars (e.g. glucose), acetoacetate, glutamine,glucosamine, N-acetyl-glucosamine (GlcNAc), and an acceptable carrier,to the individual. The invention further includes a regimen forsupplementing a healthy individual's diet to prevent a disease disclosedherein by administering daily to the human one or more of vitamin D,retinoic acid, nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP),nucleosides (e.g. cytidine or uridine), nucleobases (e.g., uracil,cytosine) sugars (e.g. glucose), acetoacetate, glutamine, glucosamine,N-acetyl-glucosamine (GlcNAc), or a nutraceutically acceptablederivative thereof.

In an aspect, the invention provides a regimen for supplementing a dietof an individual suffering form a disease disclosed herein, inparticular an autoimmune disease, more particularly MS or rheumatoidarthritis, comprising administering to the human a supplement comprisingone or more of a Vitamin D3 compound, retinoic acid, hexosamine pathwaymetabolite, nucleotides (e.g. UMP, UDP, UTP, CMP, CDP, or CTP),nucleosides (e.g. cytidine or uridine), nucleobases (e.g., uracil,cytosine) sugars (e.g. glucose), acetoacetate, glutamine, glucosamine,N-acetyl-glucosamine (GlcNAc), or nutraceutically acceptable derivativesthereof.

An individual may be treated with a supplement at least about every day,or less frequently, such as every other day or once a week. A supplementof the invention may be taken daily but consumption at lower frequency,such as several times per week or even isolated doses, may bebeneficial.

A supplement of the present invention may be ingested with or after ameal. Thus, a supplement may be taken at the time of an individual'smorning meal, and/or at the time of an individual's noontime meal. Aportion may be administered shortly before, during, or shortly after themeal. For daily consumption, a portion of the supplement may be consumedshortly before, during, or shortly after the individual's morning meal,and a second portion of the supplement may be consumed shortly before,during, or shortly after the individual's noontime meal. The morningportion and the noontime portion can each provide approximately the samequantity of one or more of a Vitamin D3 compound, retinoic acid,hexosamine pathway metabolites, nucleotides (e.g. UMP, UDP, UTP, CMP,CDP, or CTP), nucleosides (e.g. cytidine or uridine), nucleobases (e.g.,uracil, cytosine) sugars (e.g. glucose), acetoacetate, glutamine,glucosamine, N-acetyl-glucosamine (GlcNAc) or nutraceutically acceptablederivatives thereof. A supplement and regimens described herein may bemost effective when combined with a balanced diet according to generallyaccepted nutritional guidelines, and a program of modest to moderateexercise several times a week.

The compositions, supplements, and methods described herein areindicated as therapeutic or nutraceutical agents or methods either aloneor in conjunction with other therapeutic agents or other forms oftreatment. They may be combined or formulated with one or more therapiesor agents used to treat a condition described herein. Compositions ofthe invention may be administered concurrently, separately, orsequentially with other therapeutic agents or therapies. For example, acomposition or method of the invention may be used to treat MS inconjunction with β-interferon (Avonex® (interferon-beta 1a), Rebiff® (bySerono); Biogen, Betaseron® (interferon-beta 1b) Berlex, Schering),glatiramer acetate copolymer-1 (Copaxone®; Teva), antineoplastics (suchas mitoxantrone; Novatrone® Lederle Labs), human monoclonal antibodies(such as natalizumab; Antegren® Elan Corp. and Biogen Inc.),immonusuppressants (such as mycophenolate mofetil; CellCept®Hoffman-LaRoche Inc.), paclitaxel (Taxol®; Bristol-Meyers Oncology),cyclosporine (such as cyclosporin A), corticosteroids (glucocorticoids,such as prednisone and methyl prednisone), azathioprine,cyclophosphamide, methotrexate, cladribine, 4-aminopyridine andtizanidine.

A pharmaceutical pack or kit is provided comprising one or morecontainers filled with one or more of the ingredients of a compositionof the invention to provide a therapeutic effect. Associated with suchcontainer(s) can be various written materials such as labelsinstructions for use, or a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, dietary supplements, or biological products, whichnotice reflects approval by the agency of manufacture, use, or sale forhuman administration.

Polymorphism Based Treatments

The invention provides a method of treatment or prophylaxis of a diseasedisclosed herein, in particular an autoimmune disease, more particularlyMS or rheumatoid arthritis, based on the presence of a polymorphism in agene or polypeptide of the N-glycan pathway or hexosamine pathway (e.g.,SNP-containing polynucleotides and variant polypeptides).

In an aspect, the invention provides a method for treating a diseasedisclosed herein, in particular an autoimmune disease, more particularlyMS or rheumatoid arthritis, comprising obtaining a sample of biologicalmaterial containing at least one polynucleotide from the subject;analyzing the polynucleotide to detect the presence of at least onepolymorphism in a gene of the N-glycan pathway or hexosamine pathwayassociated with the disease; and treating the subject in such a way asto counteract the effect of any such polymorphism detected. In aparticular aspect, the invention provides a method for treating adisease disclosed herein, in particular an autoimmune disease, moreparticularly MS or rheumatoid arthritis, comprising obtaining a sampleof biological material containing at least one polynucleotide from thesubject; analyzing the polynucleotide to detect the presence of at leastone SNP-containing polynucleotide; and treating the subject in such away as to counteract the effect of any such polymorphism detected.

In another aspect, the invention provides a method for treating adisease disclosed herein, in particular an autoimmune disease, moreparticularly MS or rheumatoid arthritis, comprising obtaining a sampleof biological material containing at least one polypeptide from thesubject; analyzing the polypeptide to detect the presence of at leastone polymorphism in a polypeptide of the N-glycan pathway or hexosaminepathway associated with the disease; and treating the subject in such away as to counteract the effect of any such polymorphism detected. In anembodiment, the invention provides a method for treating a diseasedisclosed herein, in particular an autoimmune disease, more particularlyMS, comprising obtaining a sample of biological material containing atleast one polypeptide from the subject; analyzing the polypeptide todetect the presence of at least one polypeptide variant; and treatingthe subject in such a way as to counteract the effect of any suchpolymorphism detected.

In a particular aspect of the invention, a method is provided for theprophylactic treatment of a subject with a genetic predisposition to adisease disclosed herein, in particular an autoimmune disease, moreparticularly MS or rheumatoid arthritis, comprising obtaining a sampleof biological material containing at least one polynucleotide from thesubject; analyzing the polynucleotide to detect the presence of at leastone polymorphism in a gene of the N-glycan pathway or hexosamine pathwayassociated with the disease; and treating the subject.

In another particular aspect, the invention provides a method fortreating a disease disclosed herein, in particular an autoimmunedisease, more particularly MS or rheumatoid arthritis, comprisingobtaining a sample of biological material containing at least onepolynucleotide from the subject; analyzing the polynucleotide to detectthe presence of at least one polymorphism in a gene of the N-glycanpathway or hexosamine pathway associated with the disease; and treatingthe subject in such a way as to counteract the effect of any suchpolymorphism detected.

A subject suffering from a disease disclosed herein, ascribed to one ormore polymorphism may be treated so as to correct the genetic defect.Such a subject is identified by any method that can detect thepolymorphism(s) in a sample from the subject. A genetic defect may bepermanently corrected by administering a nucleic acid fragmentincorporating a repair sequence that supplies the wild-type nucleotideat the position of the polymorphism. A site-specific repair sequence maycomprise an RNA/DNA oligonucleotide which operates to promote endogenousrepair of a subject's genomic DNA. A site-specific repair sequence canbe administered in an appropriate vehicle, such as a complex withpolyethylenimine, encapsulated in anionic liposomes, a viral vector suchas an adenovirus, or other pharmaceutical composition that promotesintracellular uptake of the administered nucleic acid.

In cases in which a polymorphism leads to a variant polypeptide that isascribed to be the cause of, or a contributing factor to, a diseasedisclosed herein, a method of treating such a condition includesadministering to a subject a wild type cognate of the variantpolypeptide to provide complementation or remediation of thepathological condition.

Pharmacogenomics

The invention provides methods for assessing the pharmacogenomicsusceptibility of a subject harboring a particular polymorphism orhaplotype to a particular pharmaceutical compound, or to a class of suchcompounds. Pharmacogenomics relates to clinically significant hereditaryvariations, such as SNPs, in the response to therapeutics due to altereddrug disposition and abnormal action in affected subjects. The clinicaloutcomes of these variations result in severe toxicity or failure oftherapeutics in certain subjects as a result of individual variation inmetabolism. Accordingly, a SNP genotype of an individual can determinethe way a drug acts on the body or the way the body metabolizes thecompound. As an alternative to genotyping, specific variant polypeptidescan be identified. Pharmacogenomic characterization of an individualpermits the selection of effective therapeutics and effective dosagesfor prophylactic or therapeutic treatment based on the individual's SNPgenotype, thereby enhancing and optimizing the therapeutic effectivenessof the therapy. In addition, recombinant cells and transgenic animalscontaining these SNPs/haplotypes allow effective clinical design oftreatments and dosage regimens.

A polymorphism that occurs in the promoter region in the intron or is asynonymous polymorphism in the coding region is not expected to alterthe amino acid sequence of the encoded polypeptide but may affecttranscription and/or mRNA splicing, mRNA stability or translation of thesequences. Assays (e.g. reporter-based assays) may be devised to detectwhether one or more polymorphism affects transcription and/or mRNAsplicing, mRNA stability or translation. Individuals who carry allelicvariants in the promoter region, in the intron or is a synonymouspolymorphism in the coding region of a gene associated with anautoimmune disease may exhibit differences in polypeptide levels underdifferent physiological conditions and may display altered abilities toreact to disease disclosed herein, in particular an autoimmune disease.Further, differences in polypeptide levels resulting from allelicvariation may have an effect on the response of an individual to drugtherapy. For example, a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/or MGAT5polymorphisms may have an effect on the efficacy of drugs designed tomodulate the activity of a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and/orMGAT5. The polymorphisms may also affect the response to agents actingon other pathways regulated by a GCS1, GANAB, MAN1A1, MGAT1, MGAT2,and/or MGAT5. Accordingly, diagnostic methods of the invention may beuseful to assess the efficacy of therapeutic compounds in the treatmentof an autoimmune disease, predict the clinical response to a therapeuticcompound, and/or to determine therapeutic dose.

The invention also provides a method for determining the efficacy of atreatment for a particular patient with disease disclosed herein (e.g.,an autoimmune disease, in particular MS or rheumatoid arthritis), basedon genotype comprising (a) determining the genotype for one or morepolymorphism sites in a gene of the N-glycan pathway or hexosaminepathway, in particular a GCS1, GANAB, MAN1A1, MGAT1, MGAT2, and MGAT5,for a group of patients receiving a treatment; (b) sorting patients intosubgroups based on their genotype; (c) identifying correlations betweenthe subgroups and the efficacy of the treatment in the patients, (d)determining the genotype for the same polymorphism sites in the gene(s)of the particular patient and determining the efficacy of the treatmentfor the particular patient based on a comparison of the genotype withthe correlations identified in (c).

The invention further provides a method for classifying a subject who isor is not at risk for developing a disease disclosed herein (e.g. anautoimmune disease, in particular MS or rheumatoid arthritis) as acandidate for a particular course of therapy or a particular diagnosticevaluation based on a polymorphism procedure disclosed herein. Theinvention still further provides a method for selecting a clinicalcourse of therapy or a diagnostic evaluation to treat a subject who isor is not at risk for developing a disease disclosed herein (e.g. anautoimmune disease, in particular MS) based on a polymorphism proceduredisclosed herein.

Modulators of SNP-Containing Polynucleotides

The invention also contemplates a method for identifying a compound thatcan be used to treat a disease disclosed herein comprising assaying theability of the compound to modulate the activity and/or expression of aSNP-containing polynucleotide and thus identifying a compound that canbe used to treat a disease characterized by undesired activity orexpression of the SNP-containing polynucleotide. The assays can becell-based including cells naturally expressing the SNP-containingpolynucleotides or recombinant cells genetically engineered to expressthe SNP-containing polynucleotides.

The assay for SNP-containing polynucleotides can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compounds.Further, the expression of genes that are up- or down regulated inresponse to the variant SNP-containing polynucleotides can also beassayed. In this embodiment the regulatory regions of these genes can beoperably linked to a reporter gene such as luciferase.

Accordingly modulators of SNP-containing polynucleotides can beidentified in a method comprising contacting a cell with a test compoundand determining the expression of SNP-containing mRNA wherein the levelof expression of SNP-containing mRNA in the presence of the candidatecompound is compared to the level of expression of SNP-containing mRNAin the absence of the test compound. The test compound can then beidentified as a modulator of SNP-containing polynucleotide expressionbased on this comparison and be used, for example to treat a diseasecharacterized by expression of the SNP-containing polynucleotides, suchas autoimmune diseases. When expression of mRNA is statisticallysignificantly greater in the presence of the test compound than in itsabsence, the test compound is identified as an agonist of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the test compound than in its absence, thecandidate compound is identified as an antagonist of nucleic acidexpression.

The invention further provides methods of treatment, with aSNP-containing polynucleotide as a target, using a compound identifiedthrough drug screening as a gene modulator to modulate SNP-containingpolynucleotide expression. Modulation includes up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) of nucleic acid expression. Alternatively, a modulatorof SNP-containing polynucleotide nucleic acid expression can be a smallmolecule or drug identified using the screening assays described hereinas long as the drug or small molecule modulates the expression of aSNP-containing polynucleotide.

The polymorphisms of the present invention are useful for improving theprocess of drug development. Subjects can be selected for clinicaltrials based on their genotype. Individuals with genotypes that indicatethat they are most likely to respond to a treatment can be included inthe trials, and those individuals whose genotypes indicate that theywould not respond to the treatment, or suffer adverse reactions, can beeliminated from the clinical trials. In addition, the polymorphisms ofthe present invention may assist in explaining why certain, priordeveloped treatments performed poorly in clinical trials and may helpidentify a population subset that would benefit from such treatment.

The following non-limiting examples are illustrative of the presentinvention.

Example 1

Summary

Genetic and metabolic control of fractional differences in β1,61GlcNAcbranched N-glycans provide a continuum of TCR sensitivity andsusceptibility to spontaneous and induced demyelinating disease. Theencephalomyelitis (EAE) susceptible strains PL/J, SJL and NOD mice havereduced Mgat5 N-glycan expression in T-cells compared to EAE resistant129/Sv, Balb/cj and B10.S strains. Wild type PL/J mice displaying thelowest levels develop late onset spontaneous inflammatory demyelinatingdisease, which is enhanced by Mgat5^(+/−) and Mgat5^(−/−) backgrounds ina gene-dose dependent manner. Clinical disease was slowly progressive,displaying paralysis, tremor and focal dystonic posturing in associationwith axonal damage in demyelinated lesions and normal white matter;phenotypes characteristic of the progressive phase of MS (1,2). Genetargeted reduction in Mgat5 N-glycan expression linearly enhanced TCRsensitivity while increasing Mgat5 N-glycan levels by utilizingbiosynthetic precursors to raise UDP-GlcNAc supply to the Golgisuppresses T-cell proliferation, INFγ production and EAE induced in PL/Jmice by adoptive T-cell transfer.

The following methods were used in the study described in this Example.

Spontaneous Demyelinating Disease, Dystonia and Adoptive Transfer EAE.

PL/J mice at two facilities were assessed for clinical disease using astandard EAE scale (Table 1) (4). The first cohort was at backcross 4from 129/Sv (Table 1) and was housed at the Samuel Lunenfeld ResearchInstitute (SLRI) vivarium, a colony infected with mouse hepatitis virus,EDIM, Minute virus, Mouse parvovirus, GDVII, pinworm and fur mites.These mice were initially assessed by blindly examining all Mgat5^(−/−)(n=43), Mgat5^(+/−) (n=22) and Mgat5^(+/+) (n=15) PL/J mice in thecolony over 6 months of age. Only mice over 1 year of age were found tohave weakness and this smaller cohort (n=21, 13 and 10, respectively)was scored for clinical severity every 1-2 weeks over the next ˜4months. Weakness was slowly progressive without recovery in all affectedmice, an observation confirmed by daily assessment of a smaller cohort(n=12) of clinically affected mice over a 4 week period. At sacrifice,mice were perfused with paraformaldehyde via cardiac perfusion andharvested brain and spinal cord were embedded in paraffin, sectioned andstained with H&E or Luxol Fast Blue. Additional organ screening in 4clinically affected mice confirmed the only autoimmune disease presentin the mice was demyelinating disease. The second cohort (Table 3) atbackcross 6 were re-derived from the SLRI mice by embryo transfer andhoused at the University of California, Irvine (UCI) vivarium which ispathogen free except for mouse parvovirus.

Adoptive Transfer EAE.

Adoptive transfer EAE was induced by s.c. immunization of wild type PL/Jmice housed in the UCI vivarium with 100 μg of bovine MBP (Sigma)emulsified in Complete Freund's Adjuvant containing 4 mg/mlheat-inactivated Mycobacterium tuberculosis (H37 RA; Difco, Detroit,Mich.) distributed over three spots on the hind flank. Splenocytes wereharvested after 11 days and stimulated in vitro with 50 μg/ml MBP in thepresence or absence of 40 mM N-Acetyl-D-glucosamine (Sigma) added daily.After 96 hours incubation, CD3⁺ T cells were purified by negativeselection (R&D Systems) and 3.5×10⁶T-cells were injected i.p. into naïvePL/J Mgat5^(+/−) recipient mice. Trypan blue exclusion determined <3%dead cells under both culture conditions. The mice were scored daily forclinical signs over the next 30 days with the observer blinded totreatment conditions.

L-PHA Staining, Jurkat T Cells and Mouse T-Cell Proliferation.

Mice used for L-PHA staining and quantitative RT-PCR were sex and agematched. The PL/J and C57/B6 mice used were congenic at backcross 6 from129/Sv and showed no difference in L-PHA staining compared to PL/J andC57/B6 obtained from Jackson laboratories. 129/Sv mice were from anoriginal Mgat5 gene targeted population (28). All other mice (SJL, NOD,Balb/c and B10.S) were obtained from Jackson Laboratories. CD3⁺ T cellswere purified from spleens by negative selection (CD3⁺ T-cellpurification columns R&D Systems), labeled with 5 μM5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; MolecularProbes) in PBS for 8 minutes at room temperature and cultured inRPMI-1640, 10% FBS, 50 μM 2-Mercaptoethanol with plate bound anti-CD3(2C11, ebioscience) in the presence or absence of Swainsonine (Sigma)and/or N-Acetyl-D-glucosamine (Sigma). Jurkat T cells were cultured ineither RPMI 1640, 10% FBS, penicillin (200 unit/ml) and streptomycin(200 μg/ml) media at 10 mM/20 mM glucose or non-glucose containing DMEMbase media supplemented as with RPMI as well as 1.5 mM glucose, both ofwhich did not contain glutamine. The indicated monosaccharides and/ormetabolites were added daily except glucose which was added only at timezero and were titrated until a plateau was reached in L-PHA staining ortoxicity was observed. The plateau or highest non-toxic dose is shown inFIG. 4D. Mouse and Jurkat cells were stained with L-PHA and LEA by firstblocking with PBA (2% bovine serum albumin/0.01% azide/PBS) for 10 minat 4° C. and then incubated with 4 μg/ml of L-PHA-FITC (VectorLaboratories Inc.) or 20 μg/ml of LEA-FITC (Sigma) in PBA for 40 min at4° C. and analyzed by a BD FACSCalihur (BD Pharmingen, San Diego,Calif.). The level of lectin binding was normalized by untreated controland calculated as a percentage (Sample intensity/control intensity-1) %.

TCR Signaling.

10⁶ purified splenic CD3+ T cells from Mgat5^(+/+), Mgat5^(+/−) andMgat5 mice and 5×10⁶ polystyrene beads (6 micron, Polysciences) coatedwith 0.5 μg/ml anti-CD3ε antibody (2C11, eBioscience) overnight at 4° C.were mixed, pelleted at 5,000 rpm for 15 s, incubated at 37° C. for theindicated times, and then solubilized with ice-cold 50 mM Tris pH7.2,300 mM NaCl, 1.0% Triton X-100, protease inhibitor cocktail (BoehringerMannheim) and 2 mM Orthovanadate for 20 min. Cell lysates were separatedon Nupage10% BIS-TRIS gels (Invitrogen) under reducing conditions,transferred to polyvinylidene difluoride membranes and immunoblottedwith rabbit anti-phospho-lck Tyr⁵⁰⁵ Ab (Cell Signaling Technology(CST)), rabbit anti-phospho-Src family Tyr⁴¹⁶ Ab (CST), which crossreacts with phospho-lck Tyr³⁹⁴, rabbit anti-phospho-Zap70 Ab (CST),rabbit anti-phospho-LAT Ab (Upstate), and anti-actin Ab (Santa Cruz).

Cytokine ELISA.

Supernatant from spleenocyte cultures used for adoptive transfer EAE atday 4 of stimulation with MBP in the presence or absence of GlcNAc (40mM) were tested for INFγ levels. Microtiter plates were coated with 50μl of anti-IFN-γ (1 μg/ml, clone AN-18; eBiosciences) overnight at 4° C.Supernatants were applied at 50 μl/well in duplicate and incubated for 2hours at room temperature. Captured cytokines were detected usingbiotinylated anti-IFN-γ (1 μg/ml, clone R4-6A2; eBiosciences) anddetected using Avidin Horse Radish Peroxidase (eBiosciences) at 1:500×dilution and o-Phenylenediamine dihydrochloride OPD tablets (Sigma)according to the manufacturer's protocols. Recombinant IFN-γ(eBiosciences) was used as a standard. Colorimetric change was measuredat 450 nm on a microplate autoreader (Labsystems).

Quantitative Real Time PCR.

RNA from purified CD3⁺ T lymphocytes of 129/sv, PL/J and C57/B6 mice waspurified using the RNeasy® Mini Kit (Qiagen) and used to synthesize cDNAwith the RETROscript® Kit (Ambion). For expression of MGAT5 and β-actin,a 79001HT platform (3840 well plate, Applied Biosystems) was used withSYBR® Green PCR master mix and the following primers:MGAT5-5′-GGAAATGGCCTTGAAAACACA-3′ [SEQ ID NO. 1] and5′-CAAGCACACCTGGGATCCA-3′ [SEQ ID NO. 2]; for β-actin5′-CCAGCAGATGTGGATCAGCA-3′ [SEQ ID NO. 3] and 5′-TTGCGGTGCACGATGG-3′[SEQ ID NO. 4]. Automatically detected threshold cycle (Ct) values forMgat5 were normalized relative to β-actin. Fold differences inexpression were calculated based on a cDNA standard dilution curve.

MS/MS Mass Spectroscopy.

Jurkat cell pellets (20×10⁶) were resuspended in a cold 300 μlmethanol:water (1:1) solution containing maltose as internal standard,vortexed for 10 seconds, and then pipetted into tubes containing 600 μlof chloroform:methanol (C:M) (3:2). Samples were vortexed for 1 minute,and then centrifuged at 14,000 rpm for 5 minutes at 4° C. Supernatantswere collected, and an equal volume of C:M (1:1) was added, followed bya second extraction. The pooled aqueous fraction containing thehydrophilic metabolites was dried with a speedvac and stored at −80° C.Prior to injection, the samples were dissolved in 100 μl methanol:water(1:1). The samples were injected at a flow rate of 150 μl/hr into theAPI3000 Mass Spectrometer (SCIEX). The metabolites were identified bytheir transitions in MS/MS, and quantified using the Analyte Software(SCIEX), which measured the area under the curve for the fragment ionscorresponding to each parent ion. Quantities for the given substratesare graphed as pmole/min/10⁶ cells.

Electromyography and Nerve Conduction Studies.

Mice were anesthetized with Avertin and given 0.4 ml to 0.9 ml TemgesicIM for pain relief. Temperature was maintained around 35-37° C. usinginfra-red heat lamps. Monopolar needle electrodes (Ambu Inc., GlenBurnie, Md.) were used for stimulation as well for recording motor nervepotentials as well as late responses. The active recording electrode wasplaced in the medial gastrocnemius muscle with the indifferent recordingelectrode placed in the ipsilateral footpad. Active stimulatingelectrode was placed percutaneously at the popliteal region (for distalstimulation) and in the sciatic notch (proximally). Occasionally, thesciatic nerve had to be surgically exposed, using a standardized glutealsplitting approach, to optimize proximal stimulation. Referencestimulating electrode was placed in the ipsilateral thoraco-lumbarparaspinal muscle. A pre-gelled strip electrode was wrapped around thetail to act as a ground. Responses obtained from supramaximal electricalstimulation (pulse width of 0.05 msec) were analyzed; the distancebetween the stimulation sites were measured accurately using a caliper.Addition stimulations were done to record late responses (F waves and Hreflexes). H reflexes were identified when successive late responses hadidentical morphology and onset latency; F waves were identified whensuccessive late responses had variable onset latency and morphology. Forneedle EMG recording, the recording monopolar needle electrode wasinserted into one or more of the following hindlimb muscles: quadriceps,hamstrings, lumbar paraspinals, gatrocnemius and tibialis anterior.Presence of spontaneous muscle activity (i.e. fasiculations,fibrillations or myokymia) was assessed in at least three regions of themuscle. All recordings were made on a Sierra LT portable machine(Cadwell Laboratories, Kennewick, Wash.) and analyzed using theproprietary software supplied by the manufacturer.

Description of Study and Results

To further explore the role of Mgat5 N-glycans in autoimmunedemyelinating disease, the Mgat5 null allele from the EAE resistant129/Sv background was backcrossed onto the EAE sensitive PL/J strain.After the fourth backcross, signs of tail and hindlimb weakness wereobserved in mice over 1 year of age. All mice in the colony over 1 yearwere scored for severity using a standard EAE clinical scale (4) overthe next ˜4 months (Table 1). All 3 genotypes displayed limb weakness,but the incidence, severity and mortality were inversely correlated withMgat5 (Table 1). The clinical course was chronic and slowly progressivewithout relapses or recovery, a clinical picture typical of PPMS andSPMS (1,2). Clinically affected mice also displayed involuntarymovements in a gene dose dependent manner, including tremor and/or focaldystonic posturing of the tail, hindlimbs and/or spine (FIG. 1A) as wellas paroxysmal episodes of dystonia. These movement disorders are commonin patients with MS (15) but rarely reported in EAE (16). Dystonia is aneurological disorder characterized by sustained postures and twistingmovements resulting from abnormal co-contraction of agonist andantagonist muscles. Episodes of dystonia in the mice could beprecipitated by anxiety (i.e. drop from a modest height) and relieved bytouch, phenomenon typical of dystonia in humans (15).

Pathological examination revealed sub-meningeal peri-vascular lymphocytecuffing, multi-focal demyelination of the brainstem, spinal cord andspinal roots (FIG. 1B-E, FIG. 5 A-F). The CNS pathology was similar tochronic MS plaques and was characterized by mononuclear cells admixedwith myelin debris centered around blood vessels, gliosis,neuronophagia, axonal swelling (spheroids) and axonal degeneration (FIG.1C,D, FIG. 5 A,B), the latter correlating with the progressive clinicaldisease observed (17). In addition, axonal pathology was frequentlyobserved in otherwise normal appearing CNS white matter (FIG. 5C).Axonal damage has long been recognized in MS plaques (17), and morerecently in normal-appearing white matter, and is associated with theirreversible neurological deterioration in SPMS (18). The PNS pathologywas characterized by multi-focal spinal root demyelination with nakedand swollen axons (FIG. 1D, 5D-F). Neuronal bodies with prominentcentral chromatolysis were observed in the spinal cord (FIG. 5E),consistent with anterograde reaction to peripheral damage.Electromyography and nerve conduction studies revealed myokymia,positive sharp waves and delayed spinal root nerve conduction velocityas evidenced by abnormal F and H responses (FIG. 5 G,H), findingstypical of physiologic spinal root demeylination and the human PNSautoimmune demyelinating disease Chronic Inflammatory DemyelinatingPolyneuropathy (CIDP). Anti-CD3 activated splenocytes donated from Mgat5mice with moderate to severe demyelinating pathology efficientlytransferred disease to naïve wild type recipients (Table 2), confirmingspontaneous disease was autoimmune.

CNS and/or PNS pathology was present in all mice with clinical weaknessand frequently co-existed in the same individual. PNS demyelination wasseen with similar frequency in all 3 genotypes (FIG. 1F). In contrast,CNS demyelination was ˜2 and 3 fold more frequent in Mgat5^(+/+) andMgat5^(−/−) mice than wild type mice, respectively (FIG. 1F). Thisindicates that Mgat5 N-glycans specifically inhibit spontaneous CNSdemyelinating disease in a gene dose dependant manner and suggest thatthe more severe clinical disease in Mgat5^(−/−) and Mgat5^(+/−) mice wassecondary to increased frequency of CNS disease.

Spontaneous clinical and pathological disease was observed after 1 yearof age in non-congenic wildtype PL/J mice acquired from JacksonLaboratories, as well as the mice at backcross 6 to 9 (Table 2),indicating that the presence of disease in wild type PL/J mice was notsignificantly influenced by the stage of backcrossing from 129/Sv.Environmental pathogens have been suggested to promote spontaneousdemyelinating disease in MBP-TCR transgenic mice (11). In contrast,similar frequency of disease was observed when mice were housed invivariums containing only a single pathogen (Table 3) or a multitude ofpathogens (Table 1); suggesting genetic rather then environmentalfactors dominate in disease pathogenesis.

Mgat5^(+/−) mice display an intermediate incidence of autoimmunedisease, indicating Mgat5 gene dosage is limiting for modification ofN-glycans on T-cells and suppression of autoimmune activation. Indeed,flow cytometry with the Mgat5 glycan specific plant lectin L-PHA (FIGS.2A,3A) demonstrated that β1,6 branched N-glycan levels in Mgat5^(+/−)CD4⁺ T-cells are ˜30-50% lower than Mgat5^(+/−) cells in PL/J, 129/Svand C57/B6 mice (4,5) (FIGS. 2A-C, 3A and data not shown). Thetranscriptionally controlled increase in Mgat5 N-glycans following TCRstimulation (4,5) is also attenuated in Mgat5^(+/−) T-cells (FIG. 2C).Moreover, the Mgat5^(+/−) T-cell phenotype is intermediate forTCR-mediated proliferation (FIG. 2D, FIG. 6), activation of lck as shownby enhanced phosphorylation at activating tyrosine 394 (Y³⁹⁴) relativeto inhibitory tyrosine 505 (Y⁵⁰⁵)¹⁹ and phosphorylation of Zap-70 andLAT (Figure E). Therefore, a ˜30-50% reduction in Mgat5 N-glycanexpression in resting and proliferating Mgat5^(+/−) T-cells issufficient to enhance TCR sensitivity and susceptibility to spontaneousdemyelinating disease in PL/J mice.

Both PL/J and 129/Sv Mgat5^(−/−) mice develop spontaneous autoimmunedisease with pathology in different organs, notably the nervous system(Table 1) and kidneys (4), respectively. However, Mgat5-deficiencyenhances demyelinating disease in both strains (Table 1 and 4),indicating that Mgat5 regulates autoimmune thresholds irrespective ofantigen, while strain-dependent genetic factors such as MHC determinetarget antigens for spontaneous disease. Although the self-antigentargeted in PL/J mice is unknown, EAE in H-2^(μ) PL/J mice isefficiently induced with MBP, a myelin antigen expressed in central andperipheral myelin. Moreover, MBP-TCR transgenic H-2^(μ) mice developspontaneous demyelination in both the CNS and PNS (12). These datasuggest that MBP is likely the pathogenic self-antigen. Interestingly,CNS and PNS demyelination have been reported to co-exist in humanautoimmune demyelinating disease, with several studies suggestingco-occurrence in up to ˜40% of MS and CIDP patients (20-22).

Unlike the PL/J strain, Mgat5 wildtype and heterozygous 129/Sv mice donot develop spontaneous autoimmune disease, a result consistent with thelow autoimmune susceptibility of the 129/Sv strain. This differentialsensitivity to autoimmunity may in part be due to strain dependentreduction in Golgi pathway activity and expression of Mgat5-modifiedN-glycans in T-cells. Indeed, CD4⁺T⁻ cells from Mgat5^(+/+) PL/J miceexpress ˜40% less Mgat5 N-glycans then 129/Sv Mgat5^(+/+) cells, andremarkably, ˜25% less then 129/Sv Mgat5^(+/−) cells (FIG. 3A, B).Furthermore, CD4⁺ and CD8⁺ T-cells, but not B cells, from EAE highsusceptibility strains SJL and non-obese diabetic (NOD), which alsodevelops spontaneous autoimmune diabetes, expressed ˜30% less Mgat5N-glycans then the three EAF, resistant strains 129/Sv, Balb/c and B10.S(FIG. 3B). The C57/BL6 strain is less sensitive then SJL mice to activeEAE, as evidenced by differential requirement for CD28 co-stimulation(23), and displays intermediate reduction in Mgat5-modified N-glycans inT-cells. Amongst these strains, PL/J is the only one known to developspontaneous demeylinating disease, and T-cells from these mice expressedthe lowest amount of Mgat5 N-glycans without a reduction in B cells.MGAT5 mRNA expression in resting CD3⁺ T-cells from PL/J, C57/B6 and129/Sv mice are similar, indicating altered MGAT5 transcription is notresponsible for reduced Mgat5 N-glycan levels in PL/J mice (FIG. 3C).Taken together this data demonstrates an inverse relationship betweensusceptibility to autoimmune demyelinating disease and Mgat5 N-glycanexpression in T-cells with rank order PL/J>SJL, NOD>C57BL6>129/Sv,Balb/cj, B10.S and indicates that susceptible strains harbor geneticpolymorphisms that reduce Mgat5 N-glycan expression in T-cells. Since Tbut not B cell activation thresholds are regulated by Mgat5 N-glycans(4), these data strongly suggest that differences in Golgi N-glycanprocessing leading to Mgat5 N-glycans controls strain dependantautoimmune susceptibility. Indeed, TCR agonist induced phosphorylationof lck at Y³⁹⁴ and LAT in wild type PL/J T cells was significantlyenhanced relative to wild type 129/Sv T cells, while Mgat5-deficient Tcells from PL/J and 129/Sv were equally hypersensitive (FIG. 3D),confirming reduced Mgat5 glycan expression in wild type PL/J T cellsfunctions to enhance TCR sensitivity.

β1,6GlcNAc-branched products are sub-saturating on glycoproteins (FIGS.2 B,C, FIGS. 3A,B) (24), due in part to the high Km (˜10 mM) forUDP-GlcNAc displayed by the Mgat5 enzyme (25). As such, the levels ofMgat5-modified N-glycans are sensitive to changes in the intracellularconcentration of UDP-GlcNAc (26), the sugar donor synthesized de novofrom glucose via the hexosamine pathway (FIG. 4A). The addition ofGlcNAc to cultured cells can be used to supplement cellular UDP-GlcNAc,as the amino-sugar is salvaged by 6-phosphorylation and converted toUDP-GlcNAc (FIG. 4A) (26). Glutamine, Acetyl-CoA and UTP are additionalmetabolites required for UDP-GlcNAc biosynthesis, donating an amine,acetate and UDP to glucose, respectively (FIG. 4A). Supplementing humanJurkat T-cell cultures with high glucose, GlcNAc, acetoacetate,glutamine, ammonia or uridine but not control mannosamine, galactose,mannose, succinate or pyruvate dose dependently increased Mgat5N-glycans (L-PHA staining) and poly-N-acetyllactosamine (LEA staining)expression (FIGS. 4B,D, 6B). Mass spectroscopy confirmed that GlcNAc anduridine dramatically raised intracellular UDP-GlcNAc levels (FIG. 4C).Supplementing with both GlcNAc and uridine were additive for enhancementof Mgat5 N-glycans (FIG. 6 C). Supplements to wild type PL/J and C57/B6T cell cultures raised Mgat5 N-glycan expression and inhibited anti-CD3induced proliferation, an effect that could be reversed by blockingMgat5 N-glycan expression with the α-mannosidase II inhibitorswainsonine (FIGS. 4 E,F, 6D). Furthermore, re-stimulation ofsplenocytes harvested from MBP immunized wild type PL/J mice withantigen in the presence of GlcNAc doubled Mgat5 N-glycan andpoly-N-acetyllactosamine expression, inhibited INFγ production, anddramatically reduced the incidence and severity of EAE followingadoptive transfer of T-cells into naïve wildtype PL/J mice (FIGS. 4G-I).Taken together, these data confirm that reduced Mgat5 N-glycanexpression in PL/J wild type T cells functions to lower T cellactivation thresholds and enhance demyelinating disease susceptibility.Moreover, the data indicate that Mgat5 N-glycan levels and autoimmunesusceptibility are dependent on key metabolic intermediates shared byglycolysis, oxidative respiration, as well as lipid, nitrogen andnucleotide metabolism and provide a mechanism for synergism ofenvironmental and genetic factors in the promotion of autoimmunity.

Mgat5 glycans inhibit agonist induced T cell activation and T_(H)1differentiation by incorporating TCR in a galectin-glycoprotein latticethat restricts TCR clustering at the immune synapse (4,5). Here it isshown that regulation of T cell function by the Mgat5 controlled latticeis highly adaptable at both the genetic and metabolic levels, providinga molecular mechanism for tunable T cell activation thresholds (27) thatis independent of TCR ligand affinity and antigen concentration. Thecontinuous spectrum of increasing autoimmune susceptibility controlledby fractional reductions in Mgat5 glycan expression in T cells impliesthat hypomorphisms and/or environmental factors that modestly alterGolgi N-glycan and hexoamine pathway processing represent primecandidate susceptibility factors for MS and other human autoimmunediseases. The spontaneous demyelinating disease induced by Mgat5deficiency in PL/J mice phenocopies several important clinical featuresof MS; notably, spontaneous occurrence in mid-life, movement disorderssuch as tremor and dystonia and a slow progressive decline inneurological function in association with neuronal loss and axonaldamage (1, 2, 15). As such, Mgat5 deficient PL/J mice represent a uniquetool to further investigate both the inflammatory and neurodegenerativephases of MS. Mgat5 N-glycans are expressed on glycoproteins in othertissues, including neurons, where they may regulate adhesion, receptorsignaling, endocytosis, metabolism and apoptosis (24, 28, 29). Mgat5N-glycans have been shown to promote cytokine signaling, motility andphagocytosis (29), a phenotype which may also contribute to suppressionof spontaneous demyelinating disease (30). More broadly, the dataindicates a general strategy for glycotherapy, whereby proteinglycosylation can be tailored by differentially enhancingsugar-nucleotide biosynthesis with targeted mono saccharides andmetabolic precursors.

Example 2

Summary

The T cell mediated autoimmune demyelinating disease Multiple Sclerosis(MS) is a complex trait disorder, where environmental factors influencethe penetrance of mutations and complicate identification ofsusceptibility genes¹. Genetic and metabolic regulation of Mgat5modified N-glycan expression in mice sets thresholds for T cellactivation, T_(H)1 differentiation and spontaneous demyelinating diseaseby controlling clustering of T cell receptor (TCR) at the immune synapse(4,5) (Example 1). Here it is demonstrated that MS is associated withmultiple genetic deficiencies in the N-glycan pathway that reduce Mgat5glycan expression in T cells. Approximately 14/23 genes in the N-glycanand hexosamine pathways required for Mgat5 glycan expressionnon-randomly map to 18 MS and other autoimmune loci. MALDI-TOF massspectroscopy of peripheral blood mononuclear cells (PBMC) from MSpatients demonstrated high frequency blockade of N-glycan processing atthree sequential steps. Multiple single nucleotide polymorphisms (SNP)were identified in five N-glycan genes controlling the defective stepsthat were associated with disease. MS patient T cells have attenuatedup-regulation of Mgat5 glycans following TCR stimulation; a phenotypethat enhances activation, is reproduced by minimal inhibition ofproximal N-glycan processing and can be reversed metabolically withHexosamine pathway metabolites and Vitamin D3. Taken together these dataidentify genetic and environmental control of N-glycosylation as a majordefective pathway in MS and imply that a significant proportion of thegenetic heterogeneity in autoimmunity can be collapsed into genesregulating Mgat5 glycan expression. The following methods were used inthe study described in this Example.

Methods

MS patients and Control Subjects.

Patients diagnosed with Multiple Sclerosis (MS) based on the McDonaldCriteria (56) were randomly recruited from the MS clinic at theUniversity of California, Irvine. PA1,2,3,4,6,7,J represented 7 of thefirst 8 consecutive patients enrolled in the study. All were untreatedexcept for PA7, who was on low dose Methotrexate once a week and monthlycorticosteroid infusion. Four were female (PA1,3,4,J) and three weremale (PA2,6,7). Four had relapsing remitting MS (PA1,2,3,4), one hadsecondary progressive MS (PA7) and two had primary progressive MS(PA6,J). Three patients had a positive family history for MS (PA3,7,J).Controls were healthy volunteers from the University of California,Irvine campus and lacked personnel or family history of MS and otherautoimmune diseases except C2 who had a sister with autoimmune thyroiddisease.

MALDI-TOF Mass Spectroscopy.

This was done as a service at the Glycotechnology core facility, aresource of the Glycobiology Research and Training Center (GRTC) at theUniversity of California, San Diego. Resting PBMC's (6 million) werelysed with 1% SDS in 100 mM Tris pH 7.4, dialyzed to remove SDS,digested with trypsin to generate glycopeptides and then treated withPNGaseF to release the N-glycans. To focus on early processing,N-glycans were desialyated by mild acid digestion prior topermethylation and MALDI-TOF mass spectroscopy. Removal of sialic acidreduces the size and heterogeneity of the glycans, which significantlyincreases sensitivity by collapsing multiple glycans only differing intheir sialic acid content into single species with common GlcNAcbranching and galactosylation patterns. Monoisotopic peaks in thespectra were identified using GlycanMass and GlycoMod online software,with specific targeting of the N-glycan intermediates occurring duringGolgi processing.

Isolation and Activation of PBMC's.

Mononuclear cells were isolated from the blood of subjects usingHistopaque-1077™ gradients (Sigma, Saint Louis, Mo.) and cultured inRPMI 1640 medium supplemented with 4 mM glutamine, 1 mM sodium pyruvate,1% non-essential amino acids, 1% RPMI vitamins, 100 units/ml penicillin,100 μg/ml streptomycin, and 50 μM 3-mercaptoethanol. T lymphocytes wereleft unstimulated or were activated for 48 hours with 20 ng/ml anti-CD3antibody (Biomeda, Foster City, Calif.) or a combination of 20 myelinpeptides at 1 mg/ml with 10 mg/ml myelin basic protein (MBP). The myelinpeptides were immunodominant peptides from human proteolipid protein(PLP 30-49, PLP 40-60, PLP 95-118, PLP 118-150, PLP-180-189, PLP185-206), human myelin oligodendrocyte glycoprotein (MOG 38-60, MOG63-87, MOG 76-100, MOG 89-113, MOG 112-132 and MOG 162-188) and crypticpeptides from MBP (MBP 41-69, MBP 61-79, MBP 121-139 and MBP 131-149)and PLP (PLP 73-104, PLP 145-186, PLP 218-248 and PLP 241-272) that arenot normally derived from antigen processing but may be made byextracellular proteolytic activity (57). Whole MBP was isolated fromfrozen guinea pig spinal cords (Harlan Bioproducts, Indianapolis, Ind.).Cells were double-stained with PE-Cy5-conjugated anti-CD4 antibody (BDPharmingen, San Diego, Calif.) and FITC-conjugated LPHA (5). The stainedcells were analyzed on a FACScan (BD Biosciences, San Jose, Calif.)using the CellQuest software. Unstimulated cells were gated on theresting lymphoid population and the activated cells were gated on blastsbased on forward and side scatter. Background fluorescence wassubtracted for each population. To directly compare L-PHA staining inresting T cells from all subjects, PBMCs frozen in liquid nitrogen (90%FCS, 10% DMSO) were thawed and stained the same day.

Quantitative Real Time PCR and Taqman Allelic Discrimination.

Total RNA form Jurkat cell and MS patients PBMC were isolated by theRNeasy kit (Qiagen). For the time course experiments, mRNA from Jurkatcells were extracted after 0, 3, 6, 12, 24, 48, 72 hours incubation withanti-CD3 and anti-CD28 antibody (eBioscience, San Diego, Calif., USA).RNAs were reverse transcribed into cDNAs by M-MLV Reverse Transcriptase(Ambion, Inc., Austin, Tex., USA) as per the manufacture's instructions.Real-time PCR were performed on the resulting cDNAs by the use of TaqManGene Expression Assay (Applied Biosystem). Real-time PCR Primers usedwere purchased form Applied System Assays on Demand. Real-time reactionsand SNP analysis were run and analyzed by use of an ABIPRISM 7500sequence detection system and SDS2.1 software (Applied Biosystems). Andthe following cycle parameters: 50° C. for 2 minutes, 1 cycle; 95° C.for 10 minutes, 1 cycle; 95° C. for 15 seconds, 60° C. for 1 minute, 40cycles were used for real-time PCR and SNP analysis. Data were thenanalyzed with the comparative cycle threshold (CT) method. The relativemRNA expression was determined from threshold cycle values normalizedfor Actin expression and then normalized to the value derived from cellsat corresponding time without treatment.

DNA Sequencing and SNPs.

Primers designed to amplify exons of GCS1, GCS1, GANAB, MAN1A1, MGAT1and MGAT5 including ˜20-50 nucleotides of intronic sequence at both the5′ and 3′ ends were used to amplify genomic DNA isolated from PBMCs(Qiagen). Sequencing was done on gel purified PCR products (Qiagen) as aservice by Seqwright. Chromatograms and derived sequences were inspectedfor homozygous and heterozygous single nucleotide base pair changes. Allnovel SNPs were sequenced in both directions at least once, with mostbeing confirmed with a Taqman allelic discrimination assay using customdesigned probes (ABI). Introns, except for ˜20-100 nt at the 5′ and 3′end of each exon, upstream promoter regions and open boxes were notsequenced.

Novel SNPs

GCS1 SNP I: [SEQ ID NO.: 5]TGGGTATGTCGGGGCGCTGG[G/A]TGCTGGCGTGGTACCGTGCG GANAB MAN1A1 SNP X:[SEQ ID NO.: 6] GAAATAGTACAACTTAATG[G/A]ATTAGCTTTTGGGTTTAACTMGAT1 SNP VI: [SEQ ID NO.: 7]GGTGGAGTTGGTGGGTCATC[G/A]GGGCTCACTGCCTCCTGCCC MGAT5 SNP I:[SEQ ID NO.: 8] CCACTTTCTTGCTCACCTCA[C/G]CAGTTGCATGTTCTAGTCCTMGAT5 SNP II: [SEQ ID NO.: 9]GGAATCTTCTAGAAATGCCA[A/G]CTATAACCTGAAATAGTGTTKnown SNPsGCS1 SNPs: II-rs1063588, III-rs2268416. GCS1, GANAB: I-rs2957121,II-10897289, V-rs11231166. MAN1A1 SNPs: I-rs6915947, II-rs195092,III-rs9481891, IV-rs2072890, V-rs2142887, VI-rs3756943, VII-rs18513744,VIII-rs3798602, IX-rs1042800, XI-rs1046226. MGAT1 SNPs: I-rs3733751,II-rs7726357, III-rs2070924, IV-rs2070925, V-rs634501. MGAT5 SNPs:III-rs3214771, IV-rs3748900, V-rs2289465.

Since the first confirmation ˜20 years ago that MS is promoted bygenetic susceptibility (6), numerous population based whole genomescreens and candidate gene studies have failed to identify stronglylinked genes other then the MHC class II allele HLA-DRB1*15 at 6p21 (8,31-43). In Example 1 it is demonstrated that fractional reductions of30% or more in Asparagine (N)-linked glycans produced by the Golgienzyme Mgat5 promote spontaneous CNS autoimmune demyelinating disease inPL/J mice. MGAT5 encodes Golgi β1,6 N-acetylglucosaminyltransferase V,an enzyme late in the linear pathway of N-glycan branching whichgenerates the preferred intermediate for extension with polyN-acetylacsosamine (44), the high affinity ligand for galectins thatcontrols TCR sensitivity by incorporating TCR in a galectin-glycoproteinlattice (FIG. 7A) (4). The fraction of N-glycans modified by Mgat5 issensitive to donor UDP-GlcNAc levels and metabolites supplying thehexosamine pathway (Example 1, FIG. 1A). T cell hypersensitivity and EAEdemyelinating disease can be suppressed by increasing flux through thehexosamine and Golgi pathways via increased Mgat5 glycan expression(Example 1). A role for the hexosamine pathway in human autoimmunity isalso suggested by the association of a regulatory SNP near the putativeglucosamine acetyltransferase gene NAT9 (FIG. 7A, Table 4)(45).Moreover, TCR signaling increases mRNA expression of multiple upstreamN-glycan processing genes along with MGAT5, suggesting proximal N-glycanenzymes are also rate limiting for Mgat5-modified N-glycans (FIG. 7B,C).Indeed, flow cytometry with L-PHA, a plant lectin that specificallybinds to β1,6 GlcNAc branched N-glycans (FIG. 7A) (4,5), revealed dosedependent reduction of Mgat5 glycan expression in Jurkat T cellsfollowing up-stream inhibition of glucosidase I/II (GCS1/GCS1, GANAB)with castanospermine (CST), mannosidase I (MAN1A1, 1A2 and 1C1) withdeoxymannojirimycin (DMN) and mannosidase II/fix (MAN2A1, 2A2) withswainsonine (SW, FIG. 7A,C).

Based on these considerations, hyphomorphic alleles of enzymes in thehexosamine and Golgi N-glycan processing pathways leading toMgat5-modified N-glycans combine to provide a large group of potentialautoimmune susceptibility genes (FIG. 7A, Table 4). To investigate thishypothesis, linkage data for MS (8, 31, 41) and its mouse model EAE (9)were reviewed. By inclusion of genomic regions with suggestive linkageor better in three or more studies 18 potential loci covering ˜34% ofthe human genome were identified (Table 4, Table 5). The identifiedregions also frequently overlapped with other autoimmune loci (Table 4)(41, 44-50). Remarkably, 14/17 (82%) N-glycan and 11/17 (65%) hexosaminepathway genes co-localized to one of the 18 loci (Table 4 and geneslabeled blue in FIG. 7A). In contrast, 85/241 (35.3%) randomly selectedglycosylation and carbohydrate metabolic genes not known to regulateMgat5 glycan expression co-localized to the loci, a number predicted bychance given the ˜34% genome coverage of the 18 regions (Table 4, Table6). After controlling for this large percentage of the genome bysubtracting genes expected to map randomly, 13.5 of 22.5 Mgat5regulatory vs 3/159 Mgat5 non-regulatory genes co-localized to the MSassociated loci (p<0.0001, Table 4B). This result suggests that MS andother autoimmune diseases are associated with mutations in multiplegenes from the two key pathways that control Mgat5 glycan levels.

If MS patients harbor polymorphisms that lead to deficiencies inN-glycan processing, this should be detected by testing for accumulationand/or deficiencies of Golgi N-glycan intermediates by MALDI-TOF massspectroscopy (51). Analysis of the relative expression of N-glycan Golgitransients obtained from PBMC's of 5 control subjects (C1 to C5)revealed similar patterns (FIG. 8). The same analysis in 7 of 8consecutively obtained MS patients revealed significant N-glycanprocessing deficiencies in 6 of 7 patients at three distinct enzymaticsteps: Glucosidase I/II (structures A), Mannosidase I/Mgat1 (structuresB) and Mgat5 (structure G). PA1 had a dramatic reduction in glycandiversity as seen by the absence of the majority of mass peaks in thespectra relative to controls (FIG. 8B), implicating a processing blockearly in the pathway. Indeed, a marked accumulation of glycans from themost proximal steps in protein bound N-glycan processing were observed(structures A in FIG. 8) coupled with the absence of later species(structures E-G FIG. 8), suggesting mutation in GCS1 and/or GCS1, GANAB.PA3 showed a similar but less dramatic pattern, implying deficiency inthe same enzymes. In contrast, PA7 and PAJ displayed accumulation ofhigh mannose glycans (structure B in FIG. 8A), suggesting enzymaticdefects in Mannosidase I (MAN1A1, 1B1, 1C1) or Mgat1. PA2 and PA6displayed a pattern similar to controls in the proximal pathway(structures A-F), but lacked non-core-fucosylated β1,6 GlcNAc branchedtetra-antennary glycans (structure G), suggesting deficiency of MGAT5.FACS analysis with L-PHA confirmed reduced expression of Mgat5 glycansin these two patients (FIG. 10A). Remarkably, Mgat5 glycan deficientPL/J mice, a strain that develops spontaneous autoimmune demyelinatingdisease (Example 1), displayed accumulation of glycan structures at theGI/GII step (Structure A, FIG. 11A) similar to PA1 and PA3. In contrast,C57BL/6 and 129/Sv mice displayed patterns comparable to the normalhuman controls. Together, these data establish that defects in N-glycanprocessing are frequently associated with spontaneous demyelinatingdisease in humans and mice (p=0.0014, Fishers exact test).

Exons from the relevant human N-glycan genes for DNA sequencing werenext targeted (FIG. 9).

Numerous known SNPs were identified (SNPs in blue FIG. 9A), with only1/24 (PA1 MGAT5 SNP V) correlating with the glycan profiles in FIG. 8.In contrast, 7/11 previously unknown SNPs in GCS1, GCS1, GANAB, MAN1A1,MGAT1 and MGAT5 (SNPs in red FIG. 9A, 12) were predictive of glycanprocessing defects in the relevant patients (1/24 vs 7/11, p=0.0003).The absence or presence of the 8 rare correlating SNPs (cSNP, blackboxed SNPs in FIG. 9A, FIG. 11) in patients and controls (FIG. 9B,p<0.0001) as well as a comparison of the cSNPs with the other 27identified SNPs (8/8 vs 0/27, p<0.0001, Fishers exact test) were bothhighly predictive of the MALDI-TOF glycan profiles. Within the 27non-correlating SNPs, 12 were more frequent in the MS samples (ncSNPs,dashed green box FIG. 9A, 11). As the MALDI-TOF N-glycan profiles wereobtained from resting PBMCs, the ncSNPs may synergize with cSNPs duringT cell activation to attenuate the physiological increase in N-glycanprocessing (FIG. 7C, 10B,C). Indeed, the ncSNPs alone (33/154 vs 8/106,p=0.0029) or combined with the 8 cSNP (41/266 vs 8/186, p=0.0002) weredisproportionately represented in MS patient chromosomes. Moreover, thepresence of the 8 cSNPs in 5/7 MS patients or the unique cSNP/ncSNPhaplotypes in the 7 MS patients and 0 of 5 controls were bothindependently associated with MS (p=0.0278 and p=0.0013, respectively,Fishers exact test). Taken together, these data indicate cSNPs andassociated ncSNP haplotypes function to promote MS via disruption ofN-glycan biosynthesis.

To determine the significance of the genetic and biochemical block inN-glycan processing on the regulation of Mgat5 glycan expression, (β1,6GlcNAc branched N-glycans were measured by L-PHA flow cytometry. Atrest, CD4⁺ T cells from PA2 and PA6 had reduced Mgat5 glycan expression,a reduction consistent with their MALDI-TOF N-glycan profile. Mouse Tcells increase Mgat5 glycan levels ˜6-8 fold 48-72 hrs following TCRstimulation (Example 1), a fold increase that was similar to controlsC1-5 stimulated with anti-CD3 antibody or a mixture of myelin antigens(FIG. 10B). In contrast, the 7 MS patient T cells had a markedattenuation of this physiological up-regulation under both stimulatoryconditions (FIG. 10B). A second cohort of MS patients (PA8-19) andcontrols (C6-15) showed similar results, indicating dysregulation ofMgat5 glycan expression is common in MS (FIG. 10B). The defect in Mgat5glycan up-regulation was phenocopied in Jurkat and mouse T cells bytreatment with minimal concentrations of the N-glycan processinginhibitors CST, DMN or SW (FIG. 10C, 11B). The low doses usedsignificantly attenuated the up-regulation of β1,6 GlcNAc branchedN-glycans following stimulation with PMA/ionomycin or anti-CD3 withoutaffecting resting levels, a result identical to that observed in thefour MS patients—PA1, PA3, PA7 and PAJ—with cSNPs at these enzymaticsteps. Moreover, T cell proliferation is enhanced by reducing theup-regulation of Mgat5 glycans by only 30-50%, with a magnitudeequivalent to increasing TCR agonist dose ˜3-4 fold (FIG. 10D). Takentogether, these data indicate that polymorphisms which minimally alterN-glycan enzyme activity or expression are sufficient to limit theup-regulation of Mgat5 glycans and induce T cell hyperactivity. In micethis phenotype leads to preferential T_(H)1 differentiation andspontaneous autoimmune CNS demyelinating disease (5) (Example 1),implying that the genotypic and phenotypic defects in Mgat5 glycanregulation identified here directly promoted disease in each individual.

The data confirm MS is genetically heterogeneous when assessedmathematically by gene linkage analysis, but is homogeneous when genefunction is considered. The large diversity of polymorphisms in multipleN-glycan genes identified in only 7 MS patients coupled with the ˜14Mgat5 regulatory genes that non-randomly map to MS and other autoimmuneloci strongly suggest the identified SNPs represent only a smallfraction of the associated polymorphisms in the two pathways. Indeed,only 2 of the cSNPs were present in other MS patients, despite 8/10displaying attenuated up-regulation of Mgat5 glycans.

Inhibition of enzymatic activity at two separate steps in the N-glycanpathway via co-incubation of CST and SW or DMN and SW are additive inreducing Mgat5 glycan expression in Jurkat T cells (FIG. 11C),suggesting mutations in two or more genes may compound to promotedisease. This model is consistent with our observation that 6 of 7 MSpatients—PA1, PA2, PA3, PA4, PA7 and PM—had cSNPs/ncSNPs in at least twogenes in the N-glycan pathway that were absent in the C1-5 controlsamples (FIG. 7C). Moreover, 2 control subjects (C6, C9) with a singlecopy cSNP (MGAT5 SNP I and GCS1 SNP I, respectively, had normal Mgat5glycan up-regulation following TCR activation, which is in contrast toPA2, who was homozygous for MGAT5 SNP I and possessed 3 additionalncSNPs, and PA3 who had 2 cSNPs in GCS1, GANAB in addition to the GCS1SNP I (FIG. 9A,C). Furthermore, the single control subject (C10) withtwo cSNPs (homozygous MAN1A1 SNP XII FIG. 9A) had attenuated Mgat5glycan up-regulation. Together these data indicate two or more copies ofcSNPs/ncSNPs are required to produce the Mgat5 glycan phenotype.

Monozygotic twins have a ˜30% concordance rate for MS, indicating strongenvironmental influences on genetic susceptibility (6). Supplementingthe hexoasmine pathway with various metabolic intermediates common toglucose, nitrogen, lipid and nucleotide metabolism raises Mgat5 glycanlevels in T cells, inhibits proliferation, INFγ production, EAE(Example 1) and can reverse Mgat5 glycan down regulation induced byblockade of proximal N-glycan processing (FIG. 10E, 11E).1α,25-Dihydroxyvitamin D3 (Vitamin D3) up-regulates Mgat5 mRNAexpression in hepatoma cells (52), inhibits T cell activation (53), INFγproduction (54), EAE (54) and is an environmental factor controllingsusceptibility to MS (55). Addition of Vitamin D3 to Jurkat T cellsenhanced Mgat5 glycan expression, synergized with anti-CD3 inup-regulating Mgat5 glycan levels and reversed SW induced downregulationof Mgat5 glycans (FIG. 10F, 11F). Moreover, inhibition of T cellproliferation induced by Vitamin D3 is reversed with SW. Taken together,these data indicate that Vitamin D3 negatively regulates T cell functionby enhancing Mgat5 glycan expression. Thus, Vitamin D3 exposure andmetabolic flux through the hexosamine pathway provide two independentmechanisms for environmental modulation of disease promotion by theidentified SNPs and raise the possibility that genetic defects leadingto altered Mgat5 glycan expression may be relatively frequent in thenormal population but masked environmentally. Indeed, a number of normalcontrols also displayed this phenotype (FIG. 10B).

Taken together the data indicate a model where the absence or presenceof disease is dependent on the additive effects of genetic andenvironmental factors controlling flux through the hexosamine andN-glycan pathways. Moreover, treatment of MS patients with varioushexosamine pathway metabolites and/or Vitamin D alone or in combinationshould provide a simple targeted therapy to correct the underlyingbiochemical deficiency promoting disease. More broadly, the dataprovides a comprehensive approach to investigate the genetics of othercomplex trait diseases, whereby genetic heterogeneity can be simplifiedby using structural and functional analysis of entire candidatebiochemical pathways in combination with linkage data to highly focusDNA sequencing efforts.

Example 3

The following methods were used in the study described in this Example.

Methods

Spontaneous Demyelinating Disease and Dystonia.

PL/J mice at two facilities were assessed for clinical disease using astandard EAE scale (Tables 1, 3) (4). The first cohort was at backcross4 from 129/Sv (Table 1) and was housed at the Samuel Lunenfeld ResearchInstitute (SLRI) vivarium, a colony infected with mouse hepatitis virus,EDIM, Minute virus, Mouse parvovirus, GDVII, pinworm and fur mites.These mice were initially assessed by blindly examining all Mgat5^(−/−)(n=43), Mgat5^(+/−) (n=22) and Mgat5^(+/+) (n=15) PL/J mice in thecolony over 6 months of age. Only mice over 1 year of age were found tohave weakness and this smaller cohort (n=21, 13 and 10, respectively)was scored for clinical severity every 1-2 weeks over the next ˜4months. Weakness was slowly progressive without recovery in all affectedmice, an observation confirmed by daily assessment of a smaller cohort(n=12) of clinically affected mice over a 4 week period. At sacrifice,mice were perfused with paraformaldehyde via cardiac perfusion andharvested brain and spinal cord were embedded in paraffin, sectioned andstained with H&E or Luxol Fast Blue. Additional organ screening in 4clinically affected mice confirmed the only autoimmune disease presentin the mice was demyelinating disease. The second cohort (Table 3) atbackcross 6 were re-derived from the SLRI mice by embryo transfer andhoused at the University of California, Irvine (UCI) vivarium which ispathogen free except for mouse parvovirus. Disease was observed in bothpathogen-free and mouse parvovirus-containing rooms.

Adoptive Transfer EAE.

Adoptive transfer EAE was induced by s.c. immunization of wild type PL/Jmice housed in the UCI vivarium with 100 μg of bovine MBP (Sigma)emulsified in Complete Freund's Adjuvant containing 4 mg/mlheat-inactivated Mycobacterium tuberculosis (H37 RA; Difco, Detroit,Mich.) distributed over three spots on the hind flank. Splenocytes wereharvested after 11 days and stimulated in vitro with 50 μg/ml MBP in thepresence or absence of 40 mM GlcNAc (Sigma) added daily. After 96 hoursincubation, CD3⁺ T-cells were purified by negative selection (R&DSystems) and 3.6×10⁶ T-cells were injected i.p. into naïve PL/JMgat5^(+/−) recipient mice. Trypan blue exclusion determined <5% deadcells under both culture conditions. Mice were scored daily for clinicalsigns of EAE over the next 30 days with the observer blinded totreatment conditions.

FACS Analysis, L-PHA Staining and In Vitro Proliferation Assays.

Mice used for L-PHA staining and quantitative RT-PCR were sex and agematched. The PL/J and C57/BL6 mice were congenic at backcross 6 from129/Sv and showed no difference in L-PHA staining compared to PL/J andC57/BL6 obtained from Jackson Laboratories. 129/Sv mice were from theoriginal Mgat5 gene targeted population (28). All other mice (SJL, NOD,Balb/c and B10.S) were obtained from Jackson Laboratories. Mouse cellswere stained with anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD45R(RA3-6B2) from eBioscience, and L-PHA (4 μg/ml) and LEA (20 μg/ml) fromSigma. CD3⁺ T-cells (R&D Systems) were labeled with 5 μM5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; MolecularProbes) in PBS for 8 minutes at room temperature and stimulated withplate bound anti-CD3 (2C11, eBioscience) and/or anti-CD28 (37.51eBioscience) in the presence or absence of swainsonine (Sigma) GlcNAc,castanospermine (CST) and/or deoxymannojirimycin (DMN, Sigma). JurkatT-cells were cultured in either glutamine free RPMI 1640, 10% FBS, 10mM/20 mM glucose or glucose/glutamine free DMEM base media supplementedwith 10% FBS, 1.5 mM glucose. The indicated monosaccharides and/ormetabolites were added daily except glucose which was added only at timezero. Doses were titrated until a plateau was reached in L-PHA stainingor toxicity was observed. The plateau or highest non-toxic dose isshown.

TCR Signaling.

10⁶ purified splenic CD3⁺ T-cells from Mgat5^(+/+), Mgat5^(+/−) andMgat5^(−/−) mice and 5×10⁶ polystyrene beads (6 micron, Polysciences)coated with 0.5 μg/ml anti-CD3ε antibody (2C11, eBioscience) overnightat 4° C. were mixed, pelleted at 5,000 rpm for 15 s, incubated at 37° C.for the indicated times, and then solubilized with ice-cold 50 mM TrispH 7.2, 300 mM NaCl, 1.0% Triton X-100, protease inhibitor cocktail(Boehringer Mannheim) and 2 mM Orthovanadate for 20 mM. Cell lysateswere separated on Nupage10% BIS-TRIS gels (Invitrogen) under reducingconditions, transferred to polyvinylidene difluoride membranes andimmunoblotted with rabbit anti-phospho-lck Tyr⁵⁰⁵ Ab (Cell SignalingTechnology (CST)), rabbit anti-phospho-Src family Tyr⁴¹⁶ Ab (CST) whichcross reacts with phospho-lck Tyr³⁹⁴, rabbit anti-phospho-Zap70Ab (CST),rabbit anti-phospho-LAT Ab (Upstate), and anti-actin Ab (Santa Cruz).

Cytokine ELISA.

Supernatant from splenocyte cultures used for adoptive transfer EAE atday 4 of stimulation with MBP in the presence or absence of GlcNAc (40mM) were tested for IFN-γ and IL-6 levels. Microtiter plates were coatedwith 50 μl of anti-IFN-γ (1 μg/ml, clone AN-18; eBiosciences) oranti-IL-6 (1.5 μg/ml, clone MP5-20F3; eBiosciences) overnight at 4° C.Supernatants were applied at 50 μl/well in duplicate and incubated for 2hours at room temperature. Captured cytokines were detected usingbiotinylated anti-IFN-γ (1 μg/ml, clone R4-6A2; eBiosciences) oranti-IL-6 (1 μg/ml, clone MP5-32C11; eBiosciences) and detected usingAvidin Horse Radish Peroxidase (eBiosciences) at 1:500× dilution ando-Phenylenediamine dihydrochloride OPD tablets (Sigma) according to themanufacturer's protocols. Recombinant IFN-γ or IL-6 (eBiosciences) wasused as a standard. Colorimetric change was measured at 450 nm on amicroplate autoreader (Labsystems).

Quantitative Real Time PCR.

RNA from purified CD3⁺ T lymphocytes of 129/sv, PL/J and C57/BL6 mice,Jurkat I cells and transfected Lec1 cells was purified using the RNeasy®Mini Kit (Qiagen) and used to synthesize cDNA with the RETROscript® Kit(Ambion). For expression of MGAT1, 2, and 5 and β-actin, a 7900HTplatform (3840 well plate, Applied Biosystems) was used with SYBR® GreenPCR master mix and the following primers:MGAT5-5′-GGAAATGGCCTTGAAAACACA-3′ [SEQ ID NO.1] and5′-CAAGCACACCTGGGATCCA-3′ [SEQ ID NO. 2]; for β-actin 5‘-CCAGCAGATGTGGATCAGCA-3’ [SEQ ID NO. 3] and 5′-TTGCGGTGCACGATGG-3′ [SEQID NO. 4]; MGAT1-5′-CGTTGTTGGGAGATGGAAAG-3′ [SEQ ID NO. 10] and5′-TCAGGCAACAAACAAGGACA-3′ [SEQ ID NO. 11] andMGAT2-5′-AGTAGCAATGGGCGACAAAG-3′ [SEQ ID NO. 12] and5′-GCTTTGCGAAGCGAGTCTAT-3′ [SEQ ID NO.13]. For N-glycan pathway geneexpression in human Jurkat T cells, Taqman primers obtained from AppliedSystem Assays on Demand were used in the TaqMan Gene Expression Assay(Applied Biosystem). For pCMV-MGAT1 transfected LEC1 cells, thefollowing exon 2 specific primers were used with SYBR® Green PCR mastermix: 5′-CCCCGGACTTCTTCGAGTA-3′ [SEQ ID NO. 14] and5′-CGAACGTTGCCAAACTCTCT-3′ [SEQ ID NO.15]. Automatically detectedthreshold cycle (Ct) values were normalized relative to β-actin and folddifferences in expression were calculated based on a cDNA standarddilution curve.

Enzymatic assays.

Enzyme activity was measured using synthetic specific acceptors. Theacceptors for Mgat5 (GnTV), Mgat2 (GnTII) and Mgat1 (GnTI) wereβGlcNAc(1,2)βMan(1,6)βGlc-O(CH₂)₇CH₃, βGlcNAc(1,2)αMan(1,3)[αMan(1,6)]βMan-O(CH₂)₇CH₃ and αMan(1,3)βMan-O(CH₂)₇CH₃, respectively (TorontoResearch Chemicals). 10 μl cell lysate (0.9% NaCl, 1% Triton X-100 onice, centrifuged 5000 g for 15 min at 4° C.) was added to 1 mM acceptor,1 mM [6³H]-UDP-GlcNAc (Amersham) in 50 mM MES pH 6.5, 0.1 mM GlcNAc and25 mM AMP for a total reaction volume of 20 μl. Mgat2 and Mgat1reactions also contained 5 mM MnCl₂ and were incubated for 1 h; Mgat5for 3 h at 37° C. Reaction was stopped with 1 ml of ice-cold water.Enzyme products were separated from radioactive substrates by binding to50 mg C₁₈ cartridges (Alltech) preconditioned with methanol rinsing andwater washing. Reactions were loaded and the columns washed 5 times with1 ml water. Radio-labeled products were eluted directly intoscintillation vials with two separately applied 0.5 ml aliquots ofmethanol and the radioactivity was determined by liquid scintillationcounting.

MS/MS Mass Spectroscopy.

JurkaT-cell pellets (20×10⁶) were resuspended in cold 300 μlmethanol:water (1:1) solution containing maltose as an internalstandard, vortexed for 10 seconds, and then pipetted into tubescontaining 600 μl of chloroform:methanol (C:M) (3:2). Samples werevortexed for 1 minute, and then centrifuged at 14,000 rpm for 5 minutesat 4° C. Supernatants were collected, and an equal volume of C:M (1:1)was added, followed by a second extraction. The pooled aqueous fractioncontaining the hydrophilic metabolites was dried with a speedvac andstored at −80° C. Prior to injection, the samples were dissolved in 100μl methanol:water (1:1). The samples were injected at a flow rate of 150μl/hr into the API3000 Mass Spectrometer (SCIEX). The metabolites wereidentified by their transitions in MS/MS, and quantified using theAnalyte Software (SCIEX), which measured the area under the curve forthe fragment ions corresponding to each parent ion. Quantities for thegiven substrates are graphed as pmole/min/10⁶ cells.

MALDI-TOF Mass Spectroscopy.

This was done as a service at the Glycotechnology core facility, aresource of the Glycobiology Research and Training Center (GRTC) at theUniversity of California, San Diego. CD3+ murine T cells (6×10⁶),resting PBMCs from human controls and MS subjects (6×10⁶) and CHO cells(10×10⁶) were lysed with 1% SDS in 100 mM Tris pH 7.4, dialyzed toremove SDS, digested with trypsin to generate glycopeptides and treatedwith PNGaseF to release the N-glycans. To focus on early processing,N-glycans were desialylated by mild acid digestion prior topermethylation and MALDI-TOF mass spectroscopy. Monoisotopic peaks inthe spectra were identified using GlycanMass and GlycoMod onlinesoftware, with specific targeting of the N-glycan intermediatesoccurring during Golgi processing.

Carbohydrate Related Genes and MS Associated Chromosomal Regions.

β1,6GlcNAc-branched N-glycan regulatory and non-regulatory carbohydraterelated genes were identified from the literature as well as byinspection of the Kegg metabolic pathways. Additional family memberswere obtained by searches for similar gene names in the NCBI genomewebsite, resulting in identification of gene families with the same orsimilar putative function. This identified 275 carbohydrate relatedgenes (Table 6). This represents ˜1% of the predicted number of humangenes in the human genome and is consistent with previous estimates ofthe total number of carbohydrate related genes in humans (71). Thisindicates that a high percentage of human carbohydrate related geneswere incorporated in Table 4 and Table 6.

Identification of MS associated loci has been difficult with few studiesfinding significant LOD scores other than the MHC association at 6p21.Multiple groups have identified suggestive chromosomal regions but theseare often different among populations and investigators. Therefore asimple and broad approach was adopted by assuming that if three or morestudies identified a similar chromosomal cytogenetic region that thismay represent a potential MS associated region (Table 5). This approachidentified 18 regions which represented approximately 34% of the genome,the latter estimated by the proportion of nucleotides within each of the18 cytogenetic regions relative to the total as defined on the NCBIgenome website.

Electromyography and Nerve Conduction Studies.

Mice were anesthesized with Avenin. Temperature was maintained at 35-37°C. using infrared heat lamps. Monopolar needle electrodes (Ambu Inc.,Glen Burnie, Md.) were used for stimulation and recording motor nervepotentials. The active and indifferent recording electrodes were placedin medial gastrocnemius and ipsilateral footpad, respectively. Theactive and reference stimulating electrodes were placed percutaneouslyin the popliteal or sciatic notch and ipsilateral thoraco-lumbarparaspinal muscle, respectively. A pre-gelled strip electrode at thetail acted as a ground. Responses from supramaximal electricalstimulation (pulse width 0.05 msec) were analyzed with additionstimulations done to record late responses (F waves and H reflexes). Hreflexes were identified when successive late responses had identicalmorphology and onset latency; F waves were identified when successivelate responses had variable onset latency and morphology. For needle EMGrecording, the recording monopolar needle electrode was inserted intoone or more hindlimb muscles: quadriceps, hamstrings, lumbarparaspinals, gastrocnemius and tibialis anterior. The presence ofspontaneous muscle activity (i.e. fasiculations, fibrillations ormyokymia) was assessed in at least three regions of the muscle. Allrecordings were made on a Sierra LT portable machine (CadwellLaboratories, Kennewick, Wash.) and analyzed using the proprietarysoftware supplied by the manufacturer.

MS Patients and Control Subjects.

Patients diagnosed with clinically definite Multiple Sclerosis (MS)based on the McDonald Criteria (71) were randomly recruited from the MSclinic at the University of California, Irvine. PA1,2,3,4,6,7,Jrepresented 7 of the first 8 consecutive patients enrolled in the study.Both relapsing remitting and primary progressive MS patients wereincluded. Controls were healthy volunteers from the University ofCalifornia, Irvine campus and lacked personal history of MS and otherautoimmune diseases.

Isolation and Activation of PBMCs.

Mononuclear cells were isolated from the blood of subjects usingHistopaque-1077™ gradients (Sigma) and cultured in RPMI 1640 mediumsupplemented with 4 mM glutamine, 1 mM sodium pyruvate, 1% non-essentialamino acids, 1% RPMI vitamins, 100 units/ml penicillin, 100 μg/mlstreptomycin, and 50 μM β-mercaptoethanol. T lymphocytes were leftunstimulated or were activated for 48 hours with 20 ng/ml anti-CD3(Biomeda) or a combination of 20 immunodominant myelin peptides at 1mg/ml each with 10 mg/ml myelin basic protein (MBP): [human proteolipidprotein (PLP 30-49, PLP 40-60, PLP 95-118, PLP 118-150, PLP-180-189, PLP185-206), human myelin oligodendrocyte glycoprotein (MOG 38-60, MOG63-87, MOG 76-100, MOG 89-113, MOG 112-132 and MOG 162-188), and crypticpeptides from MBP (MBP 41-69, MBP61-79, MBP 121-139 and MBP 131-149),and PLP (PLP 73-104, PLP 145-186, PLP218-248 and PLP 241-272) that arenot normally derived by antigen processing but may be produced byextracellular proteolytic activity]. Whole MBP was isolated from frozenguinea pig spinal cords (Harlan Bioproducts). Cells were stained withanti-CD4 (BD Pharmingen) and L-PHA. Unstimulated cells were gated on theresting lymphoid population and the activated cells were gated on blastsbased on forward and side scatter. To directly compare L-PHA staining inresting T cells, PBMCs frozen in liquid nitrogen (90% FCS, 10% DMSO)were thawed and stained the same day.

Cloning of Human MGAT1 and Transient Transfections.

pCMV-Script PCR Cloning Kit (Stratagene) was used for human MGAT1cloning. Primers 5′-CCCCCATTTCCTCTACCTGT-3′ [SEQ ID NO.16] and5′-CCCTCCCACTCATCTGCTTTC-3′ [SEQ ID NO. 17] for human MGAT1 were used toPCR amplify exon 2 from the two exon MGAT1 gene (see FIG. 22) usinggenomic DNA of subjects with or without SNP IV/V. Exon2 contains a small5′ untranslated (UTR) region, the entire coding region and all of the 3′UTR. PCR products were sequenced and the presence or absence of SNP IV/Vwas confirmed after cloning into the pCMV vector by an allelicdiscrimination assay. 1×105 Lec1 cells (MGAT1-deficient Chinese HamsterOvary cell line) were seeded onto a 6 mm culture dish one day beforetransfection. 3 μg of plasmid DNA was diluted into 200 serum-free DMEMand 15 μl of LIPOFECTAMINE™ Reagent kit (Invitrogen) was added. Afterincubation for 30 min. at room temperature, the cells were washed oncewith 2 ml serum-free DMEM. For each transfection, 2 ml serum-free DMEMwas added to each tube containing the LIPOFECTAMINE™-DNA complexes andwere overlain onto cells. Cells were incubated for 5 hours at 37° C. ina CO₂ humidified incubator. The DNA-containing media was replaced with 2ml of DMEM supplemented with 10% FCS and cells were incubated for anadditional 72 hours. Cells were stained with L-PHA and transfectionpositive cells were determined by FACS as L-PHA-positive cells comparedto L-PHA negative vector-only transfected cells.

DNA Sequencing and SNP Analysis.

Primers designed to amplify exons of GCS1, GCS1, GANAB, MAN1A1, MGAT1and MGAT5 including ˜20-100 nucleotides (nt) of intronic sequence atboth the 5′ and 3′ ends were used to amplify genomic DNA isolated fromPBMCs using hi-fidelity Taq. Sequencing was done on gel purified PCRproducts (Qiagen) as a service by Seqwright. Chromatograms and derivedsequences were visually inspected for homozygous and heterozygous singlenucleotide base pair changes. Introns, except for ˜20-100 nt at the 5′and 3′ end of each exon, upstream promoter regions and open boxes inFIG. 22 were not sequenced. All previously unknown SNPs were sequencedin both directions at in two separate PCR reactions at least once, andconfirmed with a Tatman allelic discrimination assay using customdesigned probes (Assay by Design, Applied Biosystems). This SNP analysisassay is independent of Taq error and was run and analyzed by use of anABI PRISM 7500 sequence detection system and SDS2.1 software (AppliedBiosystems). Taqman SNP probes could not be made for MAN1A 1 IV andGCS1, GANAB SNP V and required sequencing to genotype.

Generalized Mass Action Model of Golgi N-Glycan Processing.

The Golgi N-glycan processing pathway was modeled using a series ofordinary differential equations, and generates structures up to amaximum size of tetra-antennary with N-acetyllactosamine and(Galβ1-4GlcNAcβ1-3)2 extensions. The biochemical reaction network forN-glycan processing was represented by a series of ordinary differentialequations (ODE) constructed using the generalized mass action (GMA)formulation (72). The change in concentration of each component [Ci]over time was determined by the sum of the rates of [Ci] productionminus the rates of [Ci] consumption according to the followingdifferential equation, where Ci represents one of the 143 compounds.

$\begin{matrix}{\frac{d\;\lbrack{Ci}\rbrack}{d\; t} = {{\sum{V\mspace{14mu}{production}}} - {\sum{V\mspace{14mu}{consumption}}}}} & {{Equation}\mspace{14mu}\lbrack 1\rbrack}\end{matrix}$The model includes the enzyme activities; GlcNAc-TI, II, III, IV, V,ManII, β3GlcNAc-T(i), and β4GalT, and generates structures up to amaximum size of a tetraantennary N-glycan with N-acetyllactosamine plustwo additional N-acetyllactosamine repeat extensions. The kineticproperties of these enzymes, their estimated Golgi concentrations aretaken from literature sources. The key assumptions implicit to the modelare:1) The medial and trans Golgi compartments are each considered asspatially homogeneous compartments.2) There is no loss of glycoproteins during their transport through theGolgi, and mass is conserved in the system.3) Increase in components due to cell growth is negligible, as celldoubling time is ˜20 times greater than transport rates through theGolgi.4) The following processes are considered to be linear:

-   -   a) Transit through the medial- and trans-Golgi    -   b) Glc3Man9 to Glc3Man5 N-glycan trimming in the ER and        cis-Golgi    -   c) Glycoproteins entering (protein synthesis) and exiting        (protein degradation) the system        5) Transport rates of glycoproteins to the cell surface are the        same for all glycoforms.        6) The reverse to forward (k−1/k2) ratio is assumed to be 4 for        a system operating below saturation (14) (see reaction scheme        below).

Assumptions 1 to 4a were made based on structural and biochemical datain the literature, and necessary approximations to generate an ODE modelas previously described by Umana and Bailey (74). The Umana and Baileymodel employed the Briggs-Haldane (Michaelis-Menten) approximation, acondition that is only satisfied during the initial phases of a reactionunless the amount of enzyme is very small. This situation may not bevalid for all enzymes in the Golgi at steady state. Enzyme productinhibition is significant in steps where the rate of product output isfaster than its removal. Examination of cellular N-glycan profiles inMgat5+/+ and Mgat5−/− cells by mass spectroscopy reveals that theaccumulation of the product is not limited to the step prior to Mgat5,and this is likely to hold for earlier steps in the pathway as well.Rather, product accumulation is spread through the whole pathway,suggesting extensive product inhibition. Taking the above ideas intoaccount, enzyme reactions were broken down and modeled in theirelementary forms, as outlined below. One exception was made; theBriggs-Haldane approximation was used for UDP-GlcNAc/UMP transport wherethe amount of transporter protein is much lower than the amount ofcytosolic UDP-GlcNAc and Golgi UMP (75). The transporter operates at itsVmax for most Golgi UDP-GlcNAc concentrations due to its low Km (0.00713mM), as such, establishing a direct proportionality between the steadystate amounts of UDP-GlcNAc inside the Golgi and in the cytosol. TheVmax of transport is on the order of ˜0.2 mM/s, which corresponds to themM concentration of UDP-GlcNAc in the Golgi. The GlcNAc-T enzymes (E)follow an ordered sequential bi-bi reaction mechanism beginning withbinding of the donor UDP-GlcNAc and then acceptor (A) (76). Thedissociation of UDP from the enzyme was assumed be fast, and N-glycanproduct (P) was allowed to re-bind to the enzyme.

$\begin{matrix}{{{UDP}\text{-}{GlcNAc}} + {E\underset{k - {b\; 1}}{\overset{{kb}\; 1}{ leftarrows E^{*} }}}} & \lbrack 2\rbrack \\{{A + {E^{*}\underset{k - 1}{\overset{k\; 1}{ leftarrows E^{*} }}A}}-> \overset{k\; 2}{E^{*}P}leftarrows{\underset{k - 3}{P\overset{k\; 3}{+ {UDP}}} + E} } & \lbrack 3\rbrack\end{matrix}$E*A is the activated enzyme-acceptor complex, and E*P is the activatedenzyme product complex.

Simulations of Golgi N-glycan processing were run for an arbitrary timeneeded to establish steady-state conditions and generate the N-glycanprofiles. The model allows for graded positive feedback as linearfunctions of increasing TCR activation by: (1) progressive stimulationof glucose flux through the hexosamine pathway, and (2) increasedexpression of N-glycan branching enzymes Mgat1, 2, 4 and 5, andGlcNAc-T(i), for the production of polylactosamine.

Summary

The autoimmune demyelinating disease Multiple Sclerosis (MS) resultsfrom poorly understood genetic and environmental interactions. β1,6GlcNAc-branching of N-glycans by MGAT5 titrates thresholds for T-cellactivation and autoimmunity. Here it is demonstrated MS patients andinbred mouse models have inherent genetic defects in N-glycan processingthat reduce β1,6 GlcNAc-branching and promote autoimmunity conditionalto metabolite flux through the hexosamine pathway. Defective N-glycanprocessing in PL/J mice induces spontaneous demyelinating disease.Metabolically supplementing the hexosamine pathway rescues thisphenotype and inhibits disease by increasing supply of UDP-GlcNAc toMGAT5. N-glycan and hexosamine pathway genes are over represented atputative MS loci. Glycomic analysis led to the identification of rare MSassociated single nucleotide polymorphisms (SNPs) within the N-glycanpathway. MS associated SNP IV/V doubles upstream MGAT1 activity andreduces β1-6 GlcNAc branching by limiting Golgi supply of UDP-GlcNAc.Thus, metabolic regulation of N-glycan processing by the hexosaminepathway provides a molecular intervention to reduce genetic risk todisease.

Introduction

Multiple Sclerosis (MS) is a complex trait (1) disease, whereenvironmental factors influence the penetrance of mutations andcomplicate identification of susceptibility genes. Whole genome screenshave identified a number of candidate loci associated with MS and theanimal model Experimental Autoimmune Encephalomyelitis (EAE), butnon-MHC genes with a strong association are yet to be identified (8, 9,31-41). The majority of cell surface receptors in the innate andadaptive immune responses are modified by glycosylation. TheN-glycosylation processing enzymes N-acetylglucosaminyltransferases I,II, IV and V (MGAT1, 2, 4, 5) transfer GlcNAc from the sugar nucleotidedonor UDP-GlcNAc to N-glycan intermediates transiting the medial Golgi,producing hybrid, bi-, tri- and tetra-antennary N-glycans, respectively(59) (FIG. 7A). Galectins, a family of N-acetyllactosamine-bindinglectins, bind N-glycans with increasing affinity proportional to GlcNAcbranching and poly-N-acetyllactosamine expression, the latterpreferentially expressed on β1,6GlcNAc branched tetra-antennaryN-glycans produced by MGAT5 (FIG. 7A). Galectin-3 cross-links cellsurface glycoprotein receptors (4, 29) and on T-cells, binds the T-cellreceptor (TCR) and inhibits recruitment into the immune synapse (4).Reduced β1,6GlcNAc-branching and poly-N-acetyllactosamine expression inMgat5^(−/−) T-cells induces TCR hypersensitivity and enhances T_(H)1differentiation (4, 5). Mice deficient in MGAT5 display spontaneouskidney autoimmunity and hyper-sensitivity to EAE (4).

Regulatory mechanisms controlling expression of β1,6GlcNAc-branchedN-glycans are poorly understood. The medial Golgi branching enzymesMGAT1, 2, 4 and 5 display decreasing affinities for UDP-GlcNAc, andenzyme concentrations also decline across the pathway (59) (FIG. 7A).This is consistent with observations that N-glycan structures on matureglycoproteins are sub-saturating for MGAT4 and MGAT5 products, whileMGAT1 and MGAT2 products are closer to saturation (59). Considering thehexosamine pathway supply of UDP-GlcNAc to the Golgi (FIG. 7A), hybridand bi-antennary N-glycans are produced at higher efficiency, while tri-and tetra-antennary N-glycans increase only at higher sugar-nucleotideconcentrations when the earlier enzymes become saturated. The hexosaminepathway for de novo biosynthesis of UDP-GlcNAc utilizes glucose,acetyl-CoA, glutamine and UTP (FIG. 7A), positioning UDP-GlcNAcproduction downstream of key allosteric regulators of metabolism.Finally, the majority of glucose uptake by activated T-cells isconverted to lactate and released, and it has been suggested that inaddition to energy demands, glucose may be required for growth relatedsignaling (60). Based on these considerations, it was hypothesized thatmetabolite flux into the hexosamine pathway and genetic variation inGolgi N-glycan processing efficiency may determine differences in TCRsensitivity and autoimmune susceptibility among humans and inbred mousestrains.

Results:

Incremental Differences in β1,6GlcNAc-Branched N-Glycans Titrate TCRSensitivity

Mgat5+/− T-cells from 129/Sv, PL/J and C57BL/6 mice have ˜20-25%reduction in L-PHA reactive β1,6GlcNAc branched N-glycans relative towildtype cells (FIG. 7A, 13A,B) and are intermediate compared toMgat5+/+ and Mgat5−/− cells for TCR-mediated proliferation (FIG. 13C,FIG. 17) and TCR signaling as shown by enhanced phosphorylation of lckat activating tyrosine 394 (Y³⁹⁴) relative to inhibitory tyrosine 505(Y⁵⁰⁵) and phosphorylation of Zap-70 and LAT (FIG. 13D). Therefore, theassociation of differential expression of β1,6GlcNAc-branched N-glycanswith sensitivity to EAE autoimmunity among inbred mouse strains wasexplored. Indeed, CD4⁺ and CD8⁺ T-cells, but not B cells, from the EAEhigh susceptibility strains PL/J, SJL and NOD, which also developsspontaneous autoimmune diabetes, expressed ˜30-40% lessβ1,6GlcNAc-branched N-glycans than the three EAE resistant strains129/Sv, Balb/c and B10.S (FIG. 13E). Moreover, CD4⁺ T-cells fromwildtype PL/J mice express ˜25% less β1,6GlcNAc-branched N-glycans thanMgat5+/−129/Sv cells (FIG. 13A), indicating genetic defects inherent tothe PL/J strain are significantly greater than loss of an MGAT5 allele.The C57BL/6 strain is less sensitive than SJL to induced EAE, asevidenced by differential requirement for CD28 co-stimulation (23).C57BL/6 CD8+ T-cells display intermediate levels of β1,6GlcNAc-branchedN-glycans relative to PL/J and 129/Sv cells (FIG. 13E). Therefore,susceptibility to EAE correlated inversely with β1,6GlcNAc-branchedN-glycan expression in T-cells with rank order PL/J>SJL,NOD>C57BL6>Balb/c, 129/Sv, B10.S. Indeed, PL/J T-cells were moresensitive to TCR agonist than 129/Sv T-cells as indicated byphosphorylation levels of lck at Y³⁹⁴ and LAT (FIG. 13F). In contrast,Mgat5-deficient T-cells from PL/J and 129/Sv backgrounds were equallyhypersensitive to TCR agonist, indicating that strain dependentsuppression of β1,6GlcNAc-branched N-glycans in PL/J T-cells is causalin TCR hypersensitivity (FIG. 13F).

N-Glycan Processing Efficiency Regulates β1,6GlcNAc-Branched N-Glycans

Mgat5 enzyme activity but not mRNA expression is reduced ˜50% in PL/Jand C57BL/6 splenocytes and T-cells relative to 129/Sv mice (FIG.13G,II). As PL/J T-cells have a greater reduction in β1,6GlcNAc-branchedN-glycans than C57BL/6 T-cells, the former must possess additionalN-glycan processing defects. Indeed, MALDI-TOF mass spectroscopydemonstrates that PL/J T-cells accumulate pathway intermediates upstreamof MGAT2 (i.e., E,F ions) and MGAT1 (i.e., B,C ions, FIG. 16A,B,supplementary text and FIG. 21). MGAT2 and MGAT1 enzymatic activity butnot mRNA transcript levels are reduced in PL/J>C57BL/6>129/Sv and PL/J,C57BL/6>129/Sv, respectively (FIG. 13G). An ordinary differentialequation (ODE) model of medial-Golgi enzyme reactions (materials andmethods) suggests that within the kinetic parameters of the pathway,reductions in MGAT5 and MGAT2 but not MGAT1 contribute to reducedβ1,6GlcNAc-branching in PL/J>C57BL/6>129/Sv T-cells (FIG. 17B, 15I,materials and methods). This data indicate that partial deficiencies atthe posttranscriptional level in MGAT2 and MGAT5 greater than MGAT1combine to reduce GlcNAc branching in PL/J>C57BL/6>129/Sv T-cells.However, defects in other N-glycan processing enzymes may alsocontribute to the phenotype.

β1,6GlcNAc-branched N-glycan expression is also reduced by inhibition ofglucosidase VII (GI/GII) with castanospermine (CST), mannosidase I (MI)with deoxymannojirimycin (DMN) and mannosidase II/IIx (MII/IIx) withswainsonine (SW) (FIG. 7A, FIG. 18A). Partial inhibition of enzymaticactivity at two separate steps via coincubation with CST and SW or DMNand SW are additive in reducing β1,6GlcNAc branched N-glycan expression(FIG. 18B, 19D). TCR signaling increases mRNA expression of multipleupstream N-glycan processing genes along with MGAT5, suggestingup-regulation of all processing enzymes proximal to MGAT5 are requiredto physiologically increase β1,6GlcNAc-branched N-glycan expression(4,5) (FIG. 13B, 18C). Indeed, increased expression ofβ1,6GlcNAc-branched N-glycans in activated T7 cells is attenuated withconcentrations of CST, DMN and SW that do not affect resting levels(FIG. 18D,E), indicating small decreases in N-glycan processingefficiency disproportionately reduce expression in activated T-cells.Moreover, limiting upregulation of β1,6GlcNAc-branched N-glycans by ˜50%or less is sufficient to enhance T cell proliferation (FIG. 18F). Thus,N-glycan processing efficiency is a key modulator of β1,6GlcNAc-branchedN-glycan expression in T-cells and small changes in Golgi N-glycanprocessing lead to large functional effects on TCR sensitivity andproliferation.

Metabolic Regulation of N-Glycan Processing, TCR Sensitivity and EAE

β1,6GlcNAc-branched products are sub-saturating on glycoproteins (FIG.13A,B,E) (24), due in part to the high Km (˜11 mM) for UDP-GlcNAcdisplayed by the MGAT5 enzyme. Therefore, expression ofβ1,6GlcNAc-branched N-glycans is sensitive to changes in theintracellular concentration of UDP-GlcNAc (26). Glucose, glutamine,acetyl-CoA and UTP are metabolites required by the hexosamine pathwayfor de novo UDP-GlcNAc biosynthesis (FIG. 7A). The addition ofN-acetyl-D-glucosamine (GlcNAc) to cultured cells also supplementsUDP-GlcNAc pools, following its 6-phosphorylation and conversion toUDP-GlcNAc (FIG. 7A) (26). Surface levels of β1,6GlcNAc-branchedN-glycans (L-PHA staining) and poly-N-acetyllactosamine (LEA staining)on human Jurkat T-cells were increased by supplementation with highglucose, GlcNAc, acetoacetate, glutamine, ammonia or uridine but notwith control metabolites mannosamine, galactose, mannose, succinate orpyruvate (FIG. 14A,C, 19A,B). Mass spectroscopy confirmed that GlcNAcand uridine each raised intracellular UDP-GlcNAc levels (FIG. 14B), andtheir co-supplementation displayed additive enhancement ofβ1,6GlcNAc-branched N-glycan expression and rescued proximal N-glycanprocessing insufficiency (FIG. 19C,D,E). These data demonstrate thatglucose, lipid and nitrogen metabolites are limiting for UDP-GlcNAcbiosynthesis and β1,6GlcNAc-branched N-glycans in T-cells. Moreover,they provide a molecular mechanism for metabolic modulation of GlcNAcbranching in N-glycans.

Supplements to the hexosamine pathway in wild type PL/J and C57BL/6 Tcell cultures raised Mgat5 N-glycan expression and inhibited anti-CD3induced proliferation, an effect that could be reversed by blockingβ1,6GlcNAc-branched N-glycan expression with SW (FIG. 14D-F, 19F).Furthermore, re-stimulation of splenocytes harvested from MBP immunizedwild type PL/J mice with antigen in the presence of GlcNAc increasedβ1,6GlcNAc-branched N-glycans and poly-Nacetyllactosamine expression,inhibited IFN-γ production, promoted IL-6 secretion and dramaticallyreduced the incidence and severity of EAE following adoptive transfer ofT-cells into naïve PL/J mice (FIG. 14F-H). This confirms that defectiveN-glycan processing in PL/J wild type T-cells enhances EAEsusceptibility. Moreover, the data demonstrate that the hexosaminepathway regulates TCR sensitivity and autoimmune susceptibility via keymetabolic intermediates shared by glycolysis as well as lipid (freefatty acids to acetyl-CoA), amino acid (ie ammonia/glutamine) andnucleotide metabolism.

N-Glycan Processing Defects Inherent to PL/J Mice Induce SpontaneousAutoimmune Demyelinating Disease

Although myelin-specific TCR transgenic mice develop spontaneous CNSautoimmune demyelinating disease (11-14), spontaneous disease secondaryto physiologically-relevant gene dysfunction has not been reported. Inaddition to having significant defects in N-glycan processing, PL/J micepossess the H-2μ MHC class II haplotype which limits negative selectionof MBP 1-11 reactive T-cells in the thymus (61,62). Loss of centraltolerance to MBP1-11 combined with reduced β1,6GlcNAc-branched N-glycanexpression in T-cells should induce spontaneous CNS demyelinatingdisease in PL/J mice. Indeed, clinical observation of Mgat5+/+, Mgat5+/−and Mgat5−/− PL/J mice at backcross 4 and 6 from 129/Sv as well asnon-congenic wild-type PL/J mice from Jackson Laboratories demonstratedthat all 3 genotypes displayed signs of tail and/or hindlimb weaknessafter 1 year of age (Table 1, 3, supplemental text). As predicted bydifferences in TCR sensitivity, the incidence, severity and mortalitywere inversely correlated with β1,6GlcNAc-branched N-glycan expression(Table 1, 3). The clinical course was chronic and slowly progressivewithout relapses or recovery and associated with involuntary movementssuch as tremor and focal dystonic posturing (Table 1, FIG. 20A,supplemental text), a clinical picture typical of progressive MS (1).Pathological examination revealed sub-meningeal perivascular lymphocytecuffing and multi-focal demyelination of the brainstem, spinal cord andspinal roots (FIG. 15A-C, 20B-H). The CNS pathology was similar tochronic MS plaques and characterized by mononuclear cells admixed withmyelin debris centered around blood vessels, gliosis, neuronophagia,axonal swelling (spheroids) and axonal degeneration (FIG. 15B, 17B-E,G),the latter correlating with the progressive clinical disease observed(supplemental text). Anti-CD3 antibody activated splenocytes fromMgat5−/− mice with moderate to severe demyelinating pathologyefficiently transferred disease to naïve wild type recipients (Table 2),confirming spontaneous disease was autoimmune. Raisingβ1,6GlcNAc-branched N-glycans expression in wild type PL/J T-cellsinhibits adoptive transfer EAE (FIG. 14F-H), indicating defectiveN-glycan processing in this strain is causal in promoting disease. Theseresults are the first demonstration of genetic deficiency leading tospontaneous CNS autoimmune demyelination and confirm the robustness ofsmall changes in the hexosamine and N-glycan pathways in titratingautoimmune susceptibility.

Multiple Sclerosis is Associated with Genetic Defects in N-GlycanProcessing

MS patients were examined for defects in N-glycan processing byassessing the up-regulation of β1,6GlcNAc-branched N-glycans followingTCR stimulation, which serves as a sensitive test for proximal N-glycanprocessing insufficiency (FIG. 13B, 18D,E). Murine T-cells increaseβ1,6GlcNAc-branched N-glycan levels ˜6-8 fold 48-72 hrs following TCRstimulation (FIG. 13B). A similar fold increase was observed in T-cellsfrom human controls (n=8) stimulated with anti-CD3 antibody or a mixtureof myelin antigens (FIG. 15D). In contrast, T-cells from MS patients(n=16) had a marked atenuation of this physiological up-regulation underboth stimulatory conditions (FIG. 15D), indicating defects in N-glycanprocessing occur at high frequency. Indeed, ALDI-TOF mass spectroscopyof peripheral blood monocytes (PBMCs) derived N-glycans showed 6 of 7 MSpatients had significant changes in the expression of N-glycan Golgitransients relative to controls, indicating reduced pathway output(i.e., structures A, B and G, FIG. 16A,C). Comparison with MALDI-TOFN-glycan profiles of CHO cells defective in glucosidase I (GI),mannosidase I (MI), MGAT1 or mannosidase II (MII/MIIx) confirmedN-glycan processing deficiency in MS patients and indicated defectspredominate at three enzymatic steps: Glucosidase I/II in PA1 and PA3(i.e., increased structure A), MI/MGAT1 in PA7 and PAJ (i.e., increasedstructure B) and MGAT5 in PA2 and PA6 (i.e., absence of structure G)(FIGS. 16A,C and 21 and supplemental text). These data demonstrate thatMS patients frequently display defects in N-glycan processing, aphenotype that induces spontaneous demyelinating disease in PL/J mice.

Based on these data, MS patients may harbor polymorphic alleles ofenzymes in the hexosamine and/or N-glycan pathways regulatingβ1,6GlcNAc-branching. A review of linkage data for MS (8, 31-41)obtained from 10 distinct populations as well as EAE (9) identified 18potential MS-associated chromosomal regions with suggestive linkage orbetter in three or more studies (Tables 4, 5, and 6, materials andmethods). The identified regions cover an estimated-34% of the humangenome (materials and methods) and also frequently overlap with otherautoimmune loci (Table 4) (41, 46-50). Remarkably, 14 of 17 (82%)N-glycan and 11 of 17 (65%) hexosamine pathway genes co-localized to oneof the 18 regions (Table 4 and genes labeled blue in FIG. 7A). Incontrast, 85 of 241 (35.3%) glycosylation and carbohydrate metabolicgenes not known to regulate β1,6GlcNAc-branched N-glycan expressionco-localized to the regions, a number predicted by chance given the ˜34%genome coverage of the 18 regions (Table 4B, Table 6, materials andmethods). After controlling for this large percentage of the genome,13.5 of 22.5 Mgat5 regulatory vs. 3 of 159 Mgat5 non-regulatory genesco-localized to the MS loci (Odds ratio (OR)=80.9, p<0.0001, FishersExact Test (FET), Table 2B). Thus, hexosamine and N-glycan pathway genesare significantly over represented in putative MS-associated genomicregions, supporting the hypothesis that diverse MS populations mayharbor multiple disease-associated polymorphisms in the two pathways.

To further investigate this possibility, exons of the human N-glycangenes that appeared to be defective by MALDI TOF were sequenced, namelyGCS1, GCS1, GANAB, MAN1A1, MGAT1 and MGAT5 (FIG. 22, 23). Thisidentified 24 known (blue) and 14 previously unknown (red) singlenucleotide polymorphisms (SNPs) within 5 genes from 7 MS patients (FIG.22, 23). Of these, 6 correlated with the stage of defective N-glycanprocessing observed in individual MS patients and were absent or rare incontrols (ie allele frequencies of 0%, 0%, 1.4%, 0%, 2.8% and 1.5%),suggesting they may functionally contribute to the observed phenotype(SNPs boxed in green, FIG. 22). However, altered N-glycan processing inPL/J mice results from partial deficiency of at least 2 enzymes (FIG.13G), suggesting two or more SNPs may contribute to the phenotype andpromote disease. Indeed, GANAB SNP IV (OR=2.22, p=0.015), MGAT SNP V andthe MGAT1 SNP IV/V allele (OR=17, p=0.005) are associated with disease(Table 7, FIGS. 22, 23). A large number of the SNPs were rare (greybackground FIG. 22), including 8 SNPs that were absent in controls.These rare SNPs were identified by sequencing in both directions in twoseparate PCR reactions and were confirmed multiple times in an allelicdiscrimination assay that is independent of Taq error (FIG. 22). Of theSNPs with allele frequency <5% in control subjects (SNPs with greybackground FIG. 22), 15 of 15 were more frequent in the MS cohort,having an average allele frequency of 4.2+/−0.75% in MS vs 1.0+/−0.35%in controls (p=0.0014, Mann Whitney t test). The combined chromosomalburden of these rare SNPs was associated with MS (OR=3.90, p<0.0001,Table 7). Strikingly, the presence of two or more rare SNP alleles in anindividual was highly predictive of disease (OR=44.9, p<0.0001, Table7). For comparison, the strongest autoimmune disease associatedpolymorphism in the 300 Kb region containing CD28, CTLA-4 and ICOS has aRelative Risk (RR) of 1.18 (34). Together, these data demonstrate MS isassociated with genetic polymorphisms in the N-glycan pathway andsuggest that combinations of two or more rare SNPs strongly promotedisease.

Next the function of MGAT1 SNP IV/V (FIG. 22, 23), the strongestdisease-associated allele (OR-17, p=0.005) consisting of an upstreamnon-synonymous coding polymorphism (ie SNP IV: G→A, Arg→Gln) and twosynonymous coding polymorphisms (ie SNP V: C→T (Leu→Leu) and G→T(Val→Val) 10 nucleotides apart) were assessed. Paradoxically, PBMCscontaining MGAT1 SNP IV/V have ˜2 fold increase in MGAT1 enzyme activity(FIG. 15E). In contrast, PBMC with only SNP IV had baseline MGAT1activity, suggesting the Arg→Gln change does not contribute to enhancedenzyme activity. Transient transfection of human MGAT1 with SNP IV/V orthe common allele into MGAT1 deficient CHO cells (i.e., Lec1 cells)confirmed that the SNP IV/V allele increases MGAT1 enzyme activity ˜2fold (FIG. 15F). This was associated with minimal increase in mRNAlevels (FIG. 15G), suggesting the SNP IV/V allele increases enzymeactivity at the translational level or later.

Despite enhanced enzyme activity, transfection of MGAT1 with SNP IV/Vwas ˜25% less efficient than the common allele at rescuingβ1,6GlcNAc-branched N-glycan expression in Lec1 cells (FIG. 15H). A 25%decrease in β1,6GlcNAc-branched N-glycan expression is highlysignificant as it is equivalent to being heterozygous for the MGAT5 nullallele (FIG. 13A,B) and is sufficient to enhance TCR sensitivity (FIG.13C,D,F) and susceptibility to spontaneous and induced autoimmunedemyelinating disease (Table 1, 3, FIG. 13D).

UDP-GlcNAc is limiting for β1,6GlcNAc-branched N-glycan expression (FIG.14). The MGAT1, 2, 4 and 5 GlcNAc branching enzymes display decliningaffinity for UDP-GlcNAc in rank order with Km equal to 0.04, 0.9, ˜5 and11 mM, respectively, suggesting increased MGAT1 activity may reduceβ1,6GlcNAc-branched N-glycans by limiting access of the MGAT5 enzyme toUDP-GlcNAc in the Golgi. Indeed, computational modeling of medial-Golgienzyme reactions indicated that a 2-fold increase in baseline MGAT1activity should reduce β1,6GlcNAc-branched N-glycan expression ˜35%(FIG. 15I, materials and methods, 60). Critically, hexosamine pathwaysupplementation of Lec1 cells transfected with MGAT1 SNP IV/V rescuedsurface levels of β1,6GlcNAc-branched N-glycans to that of the commonallele (FIG. 15J). These data indicate increased MGAT1 activity producedby SNP IV/V reduces β1,6 GlcNAc-branching by limiting Golgi supply ofUDP-GlcNAc. Taken together these data provide proof of principle that asin mice, genetic and metabolic interactions between the N-glycan andhexosamine pathways regulate autoimmune demyelinating disease in humans.Moreover, the data demonstrates how metabolism via the hexosaminepathway can directly influence the ability of a single human allele topromote a complex trait disease like MS.

Conclusion

The data demonstrates that genetic variability in N-glycan processingefficiency among humans and inbred mouse strains is an inherited traitthat regulates susceptibility to autoimmune demyelinating disease in amanner sensitive to metabolic flux through the hexosamine pathway.Genetic susceptibility to autoimmunity is modulated by the environmentvia unclear mechanisms. In this regard, metabolic regulation of N-glycanprocessing provides an environmental mechanism to alter inherent geneticrisk to autoimmunity. Moreover, metabolically supplementing thehexosamine pathway to increase β1,6GlcNAc-branching represents aglyco-therapeutic (65) intervention to rescue defective N-glycanprocessing in MS patients.

MS is a two stage disease characterized by T cell induced autoimmunedestruction of the myelin sheath followed by a secondary progressiveneurodegenerative phase distinguished by axonal damage and neuronal loss(31); phenotypes also present in PL/J mice. Loss of GlcNAc branchedN-glycans induces neuronal apoptosis in vivo (66), suggesting defectiveGlcNAc branching in MS patients and PL/J mice may also directlycontribute to the neurodegenerative phase of the disease. Moreover,Mgat5 deficiency inhibits macrophage motility and phagocytosis (29),which may promote autoimmune demyelinating disease by reducing clearanceof apoptotic neurons (66). β1,6GlcNAc-branched N-glycans also regulatecell adhesion, motility and endocytosis (24, 28,29), phenotypes that mayalso contribute to disease pathogenesis.

The diversity of rare polymorphisms in multiple N-glycan genesidentified in a small number of MS patients suggests that the SNPsidentified here comprise a small fraction of potentialdisease-associated polymorphisms. It appears that the rarity ofindividual SNPs is compensated for by a large number of polymorphismsdistributed over the N-glycan pathway. Furthermore, the genetic andbiochemical results provide a model whereby combinations of two or morerare SNPs in various N-glycan pathway genes are additive in reducingN-glycan processing and promoting disease. Loss of β1,6GlcNAc-branchedN-glycans induces kidney autoimmune disease in 129/Sv mice, suggestingthis model is likely relevant to a broad range of T cell mediatedautoimmune diseases. Finally, the results demonstrate an approach toidentify genes and alleles of complex trait diseases, whereby problemsof genetic heterogeneity are overcome by knowledge of a biochemicalnetwork, validation in an animal model of the disease, use of existinghuman linkage data, and robust methods predictive of molecular andcellular pathology.

MALDI TOF N-Glycan Analysis

For comparison of N-glycan profiles of mice and human MS patients, CHOcells and CHO cells deficient at 5 different steps in the N-glycanpathway (Glucosidase I, Mannosidase I, MGAT1, Mannosidase II and MGAT5,FIG. 7A) were used to define relative L-PHA and Concanavillin A (ConA)lectin staining levels by FACS along with MALDI TOF N-glycan processingprofiles. All deficiencies induced dramatic reductions inβ1,6GlcNAc-branched N-glycan expression by L-PHA staining while onlyMannosidase I, MGAT1 and Mannosidase II deficiency increasedhigh-mannose structures as seen by ConA staining (FIG. 21A). Lack ofincreased ConA staining in Glucosidase I deficient CHO cells (Lec23)⁶⁷suggests Golgi endo-mannosidase activity (68, 69), an alternativeprocessing pathway that by-passes Glucosidase I/II and allowsMannosidase I and Mannosidase II action. Indeed, a human infant withsevere human glucosidase I deficiency accumulates Glc₃Man (885 A ion inFIG. 16A, 21C) in the urine, a 4 hexose oligomer which derives from theGlucosidase I substrate Glc₃Man₉GlcNAc₂ via endo-mannosidase activity(70). MALDI TOF analysis of Lec23 cells identified a dramatic increasein a 4 hexose oligomer (885 A ion) as well as oligomers of 5 (1089) and6 (1294) residues (structure A in FIG. 16A, 21C), consistent withendo-mannosidase activity at all three mannose linkages in the α1-3mannose arm of Glc₃Man₉GlcNAc₂ (arrowheads FIG. 16A). Such activitywould produce Man₈GlcNAc₂, Man₇GlcNAc₂ and Man₆GlcNAc₂ structures andallow processing by Mannosidase I/II. Indeed, N-glycans of these 3compositions accumulate in Glucosidase I/II inhibited cells whenMannosidase I activity is blocked with DMN (69). The larger structuresGlc₃Man₉GlcNAc₂ (mass 3008) and Glc₃Man₇GlcNAc₂ (mass 2600) were alsoobserved (67), however they were at lower relative intensities comparedto the A ion group (FIG. 21C). As the amount of these ions depend on theextent of endo-mannosidase activity in the cell, which is considered lowin CHO cells (67), these data indicate analysis of A ion expression(885, 1089, 1294) provide a significantly more sensitive MALDI TOFscreening test for Glucosidase I/II deficiency when endo-mannosidaseactivity is present. MALDI TOF profiles of N-glycans from Mannosidase I(i.e., DMN treated cells) and MGAT1 (i.e., Lee 1 cells) deficient CHOcells were similar, displaying dramatic increases in high mannosestructures (i.e., structures B and C, FIG. 21D,E). In contrast,Mannosidase II (SW treated) CHO cells displayed accumulation of hybridstructures (structure D, FIG. 21F). β1,6GlcNAc-branched tetra-antennaryN-glycans (i.e., mass 2969, structure G FIG. 16A) were present in CHOcells, diminished in DMN treated cells and absent in Lec23, Lec1 and SWtreated cells. Mixing CHO and Lec1 cells in various proportionsconfirmed that relative differences in expression of N-glycan structurescan be determined using MALDI TOF (FIG. 21G).

MALDI TOF analysis of the relative expression of N-glycans obtained frommouse T cells demonstrated accumulation of the E and F N-glycanstructures in PL/J relative to C57BL/6 and 129/Sv mice (FIG. 16A,B).These structures are both directly upstream of MGAT2 (FIG. 16A) andMGAT2 enzymatic activity is decreased in PL/J>C57BL/6>129/Sv (FIG. 13G).PL/J>C57BL/6 T cells also had higher relative expression of the highmannose C and B ions that are increased in Mannosidase I and MGAT1deficient CHO cells (FIG. 16), a result consistent with MAGT1 enzymeactivity being reduced in both strains relative to 129/Sv (FIG. 13G).PL/J and C57BL/6 mice also have reduced MGAT5 activity relative to129/Sv (FIG. 13G), however, tetra-antennary β1,6 branched N-glycans(i.e., 2969, ion G) were below the level of detection in all three mousestrains. These structures were detected in human samples, suggestingspecies dependent differences in expression. Indeed, it is wellestablished that mouse T cell proliferation is induced with ConA>L-PHAwhile the opposite is true for human T cells. Indeed, direct comparisonof L-PHA staining in 129/Sv (mouse) and human T cells revealed a ˜3.5fold higher expression in the latter. Taken together, these data confirmthat PL/J T cells have significant N-glycan processing defects secondaryto multiple enzymatic deficiencies.

MALDI TOF analysis of N-glycans derived from PBMC of 5 human controlsubjects revealed similar patterns and no significant accumulation ofstructures A-G (FIG. 16A,C). The same analysis in 7 of 8 consecutivelyobtained MS patients revealed N-glycan processing deficiencies in 6 of 7patients. PA1 was similar to Lec23 cells, displaying a dramatic increasein A ions and absence of later species (i.e., ions E-G), suggestingGlucosidase I (GCS1) or Glucosidase II (GCS1, GANAB) deficiency. PA3showed a similar but less dramatic pattern, implying deficiency in thesame enzymes (FIG. 16C). PA7 and PAJ displayed accumulation of highmannose glycans (structure B in FIG. 16A,C) similar to DMN treated CHOcells and Lec1 cells, suggesting enzymatic defects in Mannosidase I(MAN1A1, 1B1, 1C1) or MGAT1. PA2 and PA6 displayed a pattern similar tocontrols in the proximal pathway (structures A-F), but lacked detectablenon-core-fucosylated β1,6 GlcNAc branched tetra-antennary glycans(structure G FIG. 16A,C), suggesting deficiency of MGAT5. Indeed, FACSanalysis with L-PHA confirmed reduced expression of β1,6GlcNAc-branchedN-glycans in resting T cells from these two patients (FIG. 21H). Thesedata indicate MS patients frequently display defects in N-glycanprocessing.

Spontaneous Demyelinating Disease in PL/J Mice

Spontaneous clinical and pathological disease were observed after 1 yearof age in non-congenic wild type PL/J mice acquired from JacksonLaboratories, as well as the mice at backcross 4 to 9 (Tables 1 and 3),indicating that the presence of disease at backcross four and six wasnot significantly influenced by the stage of backcrossing from 129/Sv.Environmental pathogens have been implicated in the promotion ofspontaneous demyelinating disease in MBP-TCR transgenic mice (II). Incontrast, similar frequency of disease was observed when mice werehoused in vivariums containing no pathogens, a single pathogen (Table 3)or a multitude of pathogens (Table 1), suggesting genetic rather thaninfectious factors dominate in disease pathogenesis.

Clinically affected mice also displayed involuntary movements in a genedose-dependent manner, including tremor and/or focal dystonic posturingof the tail, hindlimbs and/or spine (Table 1, FIG. 20A) as well asparoxysmal episodes of dystonia. These movement disorders are common inpatients with MS (15) but rarely reported in EAE. Dystonia is aneurological disorder characterized by sustained postures and twistingmovements resulting from abnormal co-contraction of agonist andantagonist muscles. Episodes of dystonia in the mice could beprecipitated by anxiety (i.e., drop from a modest height) and relievedby touch, phenomena typical of dystonia in humans (15).

Axonal pathology was frequently observed in otherwise normal appearingCNS white matter (FIG. 17E). Axonal damage has long been recognized inMS plaques, more recently in normal-appearing white matter, and isassociated with the irreversible neurological deterioration in SPMS².The PNS pathology was characterized by multi-focal spinal motdemyelination with naked and swollen axons (FIG. 15C, 20F,H). Neuronalbodies with prominent central chromatolysis were observed in the spinalcord (FIG. 20G), consistent with anterograde reaction to peripheraldamage. Electromyography and nerve conduction studies revealed myokymia,positive sharp waves and delayed spinal root nerve conduction velocityas evidenced by abnormal F and H responses (FIG. 20I,J), findingstypical of physiologic spinal root demyelination and the human PNSautoimmune demyelinating disease, Chronic Inflammatory DemyelinatingPolyneuropathy (CIDP).

CNS and/or PNS pathology was present in all mice with clinical weaknessand frequently co-existed in the same individual. PNS demyelination wasseen with similar frequency in all 3 Mgat5 genotypes (FIG. 20K). Incontrast, CNS demyelination was ˜2 and 3 fold more frequent inMgat5^(+/−) and Mgat5^(−/−) mice than wild type mice, respectively (FIG.20K). This indicates that β1,6GlcNAc-branched N-glycans d N-glycanssuppress spontaneous CNS demyelinating disease in a gene dose dependentmanner and suggests the more severe clinical disease in Mgat5^(−/−) andMgat5^(+/−) mice was secondary to increased frequency of CNSdemyelination.

The spontaneous demyelinating disease induced by Mgat5 deficiency inPL/J mice phenocopies several important clinical features of MS:spontaneous occurrence in mid-life, movement disorders such as tremorand dystonia and a slow progressive decline in neurological function inassociation with neuronal loss and axonal damage (2,15). As such,Mgat5-deficient PL/J mice represent a unique model to study both theinflammatory and neurodegenerative phases of MS.

Example 4

To determine whether MGAT1 SNP V is associated with other autoimmunediseases in addition to MS patients were analyzed with RheumatoidArthritis (RA) and Thyroid Autoimmunity (ie Graves disease andHashimoto's) as well as MS and control cohorts were expanded (Table 7).Case control analysis demonstrated that both MS and RA are associatedwith disease with similar odds ratio's, while Thyroid Autoimmunity haslittle or no association. The transmission disequilibrium test (TDT), afamily based test of association that eliminates bias from populationselection, confirmed MS and RA but not Thyroid Autoimmunity areassociated with MGAT1 SNP V.

Example 5

Low concentrations of Glucosamine increase Mgat5-modified N-glycanexpression in Jurkat T while high concentrations do the opposite (FIG.24 A,B). As glucosamine but not GlcNAc also competes with glucose forthe glucose transporter, may promote insulin resistance and can beconverted to Fructose-6-phosphate to enter glycolysis (FIG. 24A), GlcNAcis a preferred therapeutic. However, due to its lack of hydrophobicity,GlcNAc enters the cell poorly. This can be significantly improved byacetylating GlcNAc (ie GlcNAc-tetra-acetate) to increase hydrophobicityand cell entry. Cytoplasmic de-acetylases will remove the acetyl groupsto produce GlcNAc following cell entry. Indeed, GlcNAc-tetra-acetate isable to increase Mgat5-modified N-glycan expression at effectiveconcentrations ˜1000 fold less than GlcNAc (FIG. 24 A). Similarly,removal of the hydrophilic ribose from uridine to form the base uracilsignificantly increases hydrophobicity and reduces the effectiveconcentration required to raise Mgat5-modified N-glycan expression ˜100fold compared to uridine (FIG. 24 C).

Vitamin D3 increases Mgat5-modified glycan expression by increasing mRNAexpression of Mgat5 and possibly other N-glycan and hexosamine pathwaygenes while hexosamine pathway supplementation increases Mgat5 modifiedN-glycan expression by increasing UDP-GlcNAc, the sugar nucleotide donorfor Mgat1 1, 2, 4 and 5. As these are two independent mechanisms toraise Mgat5-modified N-glycan expression, combining these should besynergistic. Indeed, Vitamin D3 coupled with hexosamine pathwaysupplementation (ie GlcNAc-tetra-acetate and uridine) synergisticallyincreased Mgat5-modified N-glycan expression. Thus, this combination maybe particularly useful to therapeutically raise Mgat5-modified N-glycanexpression in autoimmune disease patients (eg MS and RA). Combinationtherapy utilizing multiple hexosamine pathway metabolites and/or VitaminD3 will also ensure that supply of metabolites is not limiting as wellas minimize the concentration of individual therapeutics and therebylimit potential toxicity and negative feedback. shows that acetylatedGlcNAc and uracil increase Mgat5 glycan expression at ˜100-1000 foldlower concentrations than GlcNAc and uridine. The new data also showsthat Glucosamine works at low concentrations but then declines at laterconcentrations, which does not occur with the other supplements. ThusGlcNAc is a better agent than glucosamine. The data also show thatVitamin D3 plus GlcNAc+uridine are synergistic in increasing Mgat5modified N-glycans.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. All publications, patents and patent applicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the molecules, methodologies etc. which arereported therein which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anagonist” includes a plurality of such agonists, and equivalents thereofknown to those skilled in the art, and so forth.

TABLE 1 Table 1: Clinical Observations of Spontaneous Disease In PL/Jmice Weakness Dystonia Genotype n Age(m) Incidence(%)* Score^(#)Incidence(%)^(§) Score^(†) Death(%)^(¥) Mgat5^(+/+) 10   14 +/− 0.4 200.4 +/− 0.27 0 0   — 17.5 +/− 0.6 40 0.8 +/− 0.33 10 0.1 0 Mgat5^(+/−)13 15.6 +/− 0.7 38.5 0.8 +/− 0.28 23.1 0.69 +/− 0.37 — 18.2 +/− 0.8 69.21.6 +/− 0.38 38.5 0.92 +/− 0.37 7.7 Mgat5^(−/−) 21 15.4 +/− 0.5 61.9 1.3+/− 0.25 42.9 0.86 +/− 0.26 — 18.2 +/− 0.4 81.0 2.9 +/− 0.44 52.4 1.10+/− 0.28 38.1 Severity of weakness was scored on a scale of 0-5 with: 0,no weakness; 1, limp tail; 2, hindlimb weakness; 3, hindlimb paralysis;4, forelimb weakness/paralysis and hindlimb paralysis; and 5,moribundity or death. Severity of dystonia was scored on a scale of 0-3with: 0, no dystonia, 1, tail dystonia; 2, hindlimb dystonia; 3, axialskeleton and/or paroxysmal dystonia. Time and age are given in months.Percentages for spontaneous death and incidence were cumulative.Severity is mean +/− standard error. n was the denominator for allcalculations. *p = 0.0234 (0 m), p = 0.0263 (4 m) chi square test fortrend comparing all 3 genotypes ^(#)p = 0.0007; Two Way Anova comparingall 3 genotypes at both time points ^(§)p = 0.04 chi square (time 0); p= 0.0263 (4 m) chi square test for trend comparing all 3 genotypes ^(†)p= 0.0076; Two Way Anova comparing all 3 genotypes at both time points^(¥)p = 0.0194 chi square comparing all 3 genotypes

TABLE 2 Adoptive Transfer of Demyelinating Disease into wildtype PL/Jmice Donor Recipient Genotype Severity Incidence* Onset(w) Score^(#)Pathology Mgat5^(−/−) + 0/6 0 0 0/6 Mgat5^(+/−) ++/+++ 1/6 3 0.33 +/−0.33 1/4 Mgat5^(−/−) ++/+++ 3/3 3 +/− 1.3 3.0 +/− 1.0 3/3 Spontaneouslydiseased donor mice were scored for severity of demyelinating pathology(+ to +++) and harvested splenocytes were stimulated 48 hrs in vitrowith anti-CD3 + anti-CD28 antibody and then injected ip into naivewildtype PL/J mice. The recipient mice were scored for weakness andpresence of demyelinating pathology. Score is mean +/− standard error. w= weeks *p = 0.0114, chi square comparing all 3 genotypes ^(#)p =0.0056; One Way Anova (Kruskal-Wallis)

TABLE 3 Clinical Observations of Spontaneous Disease in PL/J mice at UCIGenotype n Age(m) Incidence(%) Score Death(%) Mgat5^(+/−) 27 13.7 ± 0.23.70 0.19 ± 0.19 — 25 15.6 ± 0.2 16.0 0.72 ± 0.34 7.4 22 17.5 ± 0.2 59.12.05 ± 0.40 18.5 Mgat5^(−/−) 17 13.0 ± 0.4 11.8 0.59 ± 0.40 — 14 15.0 ±0.4 35.7 1.36 ± 0.53 17.7 8 16.3 ± 0.5 87.5 3.13 ± 0.55 53.0 PL/J miceat backcross six from 129/Sv housed in the University of California,Irvine (UCI) vivarium were examined twice weekly for clinical signs ofspontaneous demyelinating disease and scored in a blinded fashion asdescribed in Table 1. Data are tabulated at 3 separate time points over4 months. Percentages for Death and Incidence are cumulative. Data arepresented as mean ± s.e.m.

TABLE 4 β1,6GlcNAc-branched N-glycan regulatory genes and 18 putative MSloci A) Autoimmune Loci β1,6GlcNAc-branched N-glycan regulatory genes MSOther^(#) Gene Location Enzyme 1p21-p36 IBD, SLE, EAE DDOST 1p36.1Oligodolichol Transferase MAN1C1 1p35-p36 Golgi Mannosidase IC SLC35A31p21 UDP-GlcNAc Tansporter, Golgi 1q11-q45 IDDM, PS, SLE UAP1 1q23UDP-GlcNAc Pyrophosphorylase 1 B4GALT3 1q21-q23 GlcNAc β1,4galactosyltransferase III 2q14-q32 — MGAT5 2q21-q22 N-AcetylglucosaminylTransferase V 2q36-q37 SLE — — — 2p11-24 IDDM, AITD, EAE GCS1 2p12-p13Glucosidase I GFPT1 2p13 Fructose-6-PO4-transaminase 1 NAGK 2p13N-acetylglucosamine kinase HK2 2p13 Hexokinase 2 4q28-35 PS, EAE — — —5p12-p15 SLE, EAE — — — 5q14-q35 IDDM, PS, IBD MAN2A1 5q21-q23Mannosidase II AITD, EAE MGAT1 5q35 N-Acetylglucosaminyl Transferase IMGAT4b 5q35 N-Acetylglucosaminyl Transferase IVb GNPDA 5q21Glucosamine-6-PO4-deaminase 1 GFPT2 5q34-q35 Fructose-6-PO4-transaminase1 HK3 5q35.2 Hexokinase 3 6q22-q27 IDDM, PS MAN1A1 6q22 Mannosidase Ia10p12-p15 — — — — 11p13-p15 IDDM — — — 11q12-q15 IDDM, AITD, EAE GANAB11q12 Glucosidase II B3GNT6 11q13.2 β1,3-GlcNAc Transferase VI (IGnT)12q21-q24 IBD, AITD B3GNT4 12q24 β1,3-GlcNAc Transferase IV 14q21-q32IDDM, PS, AITD MGAT2 14q21 N-Acetylglucosaminyl Transferase II GNPNAT114q22.1 Glucosamine-6-PO4-N-acetyltransferase 16p12-p13 IDDM, EAE — — —17q11-q25 IDDM, IA, PS, EAE NAT9 17q25Glucosamine-6-PO4-N-acetyltransferase 19q12-q13 IA, SLE GPI 19q13Glucose Phosphate Isomerase 22q12-q13 EAE MGAT3 22q13.1N-Acetylglucosaminyl Transferase III B) Map to 18 MS Loci CarbohydrateGenes Observed* Expected Observed-Expected^(#) β1,6GlcNAc Non-regulatory85/241 (35.3%) 82/241 (34%) 3/159 (1.9%) β1,6GlcNAc Regulatory 25/34(73.5%) 11.5/34 (34%) 13.5/22.5 (60%) *,^(#)p < 0.0001, Fishers Exacttest, # Relative Risk 32.3, Odds Ratio 80.9 MS loci are defined by theirsuggestive or better association/linkage (ie LOD >1) to MS/EAE in atleast 3 of 12 studies shown in Table S3, excluding the known MHCassociation at 6p21. These cytogentic regions account for ~34% of thehuman genome (materials and methods), a number controlled for in B.Other autoimmune loci are shown if they co-localize to the MS loci.β1,6GlcNAc-branched N-glycan regulatory and non-regulatory genes areshown in FIG. 1 and Table S4, respectively. Non-regulatory genes controlmetabolism of other carbohydrates. IDDM: autoimmune diabetes. IBD:inflammatory bowel disease, SLE: systemic lupus erythematosis. PS:psoriasis. AITD: autoimmune thyroid disease. IA: inflammatory arthritis.EAE: experimental autoimmune encephalomyelitis.

TABLE 5 MS and EAE linked/associated loci Canada USA/France BritainItalian Finland Sweden Norway (Dyment (Pericak-Vance (Sawcer (Broadley(Kuokkanen (Giedraitis (Akkesson Loci et al) et al) et al) et al) et al)et al) et al) 1p21-p36 1p31-36 1p34 1p34 — — — — 1q11-q45 1q24-q25 —1q43 1q25-1q44 — — 1q11-q24 2q14-q32 2q21-22 — — — — 2q14-q31 2q24-q322q36-q37 2q37-qtel — — 2q36 — — — 2p11-p24 — — 2p21 — — — — 4q28-35 4q32— — — — — — 5p12-p15 5p15-ptel — — — 5p12 — — 5q14-q35 — — — 5q33 —5q22-q23 — 6q22-q27 6q27 — — 6q22-25 — 6q25-q27 — 10p12-p15 10p15 — —10p11-cen — — 10p15 11p13-p15 11p13-p15 — — — — — 11p15 11a12-q15 — — —— — — — 12q21-q24 12q21-q23 — — — — — 12q21 14q21-q32 — — — — —14q24-q32 — 16p12-p13 — — — — — 16p12-p13 16p13 17q11-q25 17q21-q23 —17q23-q24 — 17q22-q24 17q12-q24 17q25 19q12-q13 — 19q13 19q13.3 — — — —22q12-q13 — — — — — — 22q12-q13 Sardinian Australian GAMES Becker EAELoci (Coraddu et al) (Ban et al) (2003) et al (Butterfield et al)1p21-p36 — — — 1p21-p36 1p12-p31 1q11-q45 — — — — — 2q14-q32 — — — — —2q36-q37 2q36 — — — — 2p11-p24 — — 2p14 2p11-p24 2p12-p23 4q28-q35 — — —4q28-q35 4q28-q33 5p12-p15 — — — 5p16 5p13-p15 5q14-q35 — — — 5q145q31-p35 6q22-q27 — — — — — 10p12-p15 10p12 — 10p15 — — 11p13-p15 — —11ptr — — 11a12-q15 — 11q12 — 11q14 1q13-q15 12q21-q24 — — — 12q21-q24 —14q21-q32 — 14q21 — 14q32 — 16p12-p13 — 16p13 16p13 16p13 16p1317q11-q25 — — 17q21 17q21-q23 17q11-23 19q12-q13 — 19q13 — 19q12-q13 —22q12-q13 — — 22q13 22q13 22q12

TABLE 6 Carbohydrate genes not required for β1,6GlcNAc-branched N-glycansynthesis Gene Location Enzyme Glycan type A) Genes that Map to 18 MSLoci in Table 5 B3GNT1 2p15 β1,3 N-Acetylglucosaminyltransferase I GAG,Glycolipid B3GNT7 2q37.1 β1,3 N-Acetylglucosaminyltransferase VII ?B4GALT2 1p33-p34 β1,4 Galactosyltransferase II N-Glycans, GlycolipidB4GALT7 5q35 β1,4 Galactosyltransferase VII Proteoglycans B3GALT1 2q24.3β1,3 Galactosyltransferase I Glycolipid B3GALT2 1q31 β1,3Galactosyltransferase II N-,O-Glycan B3GALT6 1q36 β1,3Galactosyltransferase VI N-,O-Glycan B3GALT7 19q13 β1,3Galactosyltransferase VII N-,O-Glycan GALNT2 1q41-q42N-Acetylgalactosaminyltransferase II O-Glycans GALNT3 2q24-q31N-Acetylgalactosaminyltransferase III O-Glycans GALNT4 12q21.3-q22N-Acetylgalactosaminyltransferase IV O-Glycans GALNT5 2q24.2N-Acetylgalactosaminyltransferase V O-Glycans GALNT7 4q31.1N-Acetylgalactosaminyltransferase VII O-Glycans GALNT9 12q24.3N-Acetylgalactosaminyltransferase IX O-Glycans GALNT10 5q33.2N-Acetylgalactosaminyltransferase X O-Glycans GALNT13 2q24.1N-Acetylgalactosaminyltransferase XIII O-Glycans GALNT14 2p23.2N-Acetylgalactosaminyltransferase XIV O-Glycans GALGT2 17q21.33N-Acetylgalactosaminyltransferase Blood Group Antigen SIAT6 1p34.1 α2,3Siayltransferase (ST3Gal III) N-,O-Glycans, Glycolipid SIAT7A & B17q25.3 α2,6 Siayltransferase (ST6GalNAc I) O-Glycan SIAT7C & E 1p31.1α2,6 Siayltransferase (ST6GalNAc III) O-glycan, Glycolipid SIAT8D 5q21α2,8 Siayltransferase IV (ST8Sia IV) N-glycan SIAT9 2p11.2 α2,3SiayltransferaseV (ST3Gal V) Glycolipid FUT1, 2 & 3 19q13.3 α1,2Fucosyltransferase ? FUT5 & 6 19q13.3 α1,3 Fucosyltransferase ? FUT814q24.3 α1,6 Fucosyltransferase N-Glycan DPM3 1q22Dol-P-mannosyltransferase 3 GPI-linked LOC402458 11p15.4 Similar to β1-4mannosyltransferase ? LOC399914 11q13.1 Similar to β1-4mannosyltransferase ? LOC401658 11p15.5 Similar to β1-4mannosyltransferase ? HS3ST2 16p12 Heparin Sulfate 3-O-sulfotransferaseII GAG HS3ST6 16p13.3 Heparin Sulfate 3-O-sulfotransferase VI GAG HS2ST11p22-p31 Heparan Sulfate 2-O-sulfotransferase GAG HS6ST1 2q21 HepranSulfate6-O-sulfotransferase GAG CHST8 19q13.1N-Acetylgalactoasaminyltransferase-4-O-Sulfotransferase VIII GAG CHST1112q24 Chondroitin-4-O-Sulfotransferase GAG UST 6q24Uronyl-2-sulfotransferase GAG NDST1 5q33N-deacetylase/N-sulfotransferase I GAG EXTL1 1p36.1 α1,4 GlcNActransferase GAG EXTL2 1p21 α1,4 GlcNAc transferase GAG B3GAT3 11q12.3β1,3 glucuronyltransferase III GAG XYLT2 17q21-q22 Xylosetransferase IIGAG NAGLU 17q21 α-N-acetylglucosaminidase GAG CTBS 1p22 Chitobiase ?NEU2 2q37 Sialidase cytosolic sialidase NEU3 11q13 Sialidase N-glycandegradation NEU4 2q37.3 Sialidase N-glycan degredation GBA 1q21Glucosidase B Glycolipid GAA 17q25 Glucosidase (Pompes Disease) Glycandegradation FLJ21865 17q25.3 N-aetylglucosaminidase N-glycan degradationFUCA1 1p34 Fucosidase N-glycan degradation UGP1 1q21-q22 UDP-glucosepyrophosphorylase 1 Sugar-nucleotide UGP2 2p13-p14 UDP-glucosepyrophosphorylase 2 Sugar-nucleotide GMPPA 2q36.1 GDP-mannosepyrophosphorylase Sugar-nucleotide PGM1 1p31 Phosphoglucomutase 1Glycolysis ACYP1 14q24 Acylphosphatase 1 Gluconeogenesis ACYP2 2p16.2Acylphosphatase 2 Gluconeogenesis ENO1 1p36 Enolase 1 Glycolysis PKLR1q21 Pyruvate Kinase Gluconeogenesis PFKP 10p15Phosphofructokinase-platelet Glycolysis LDHA 11p15 Lactate dehydrogenaseA Glycolysis AKR1A1 1p32-p33 Aldehyde reductase A1 Glycolysis ASML3B1p35 Sphingomyelin phosphodiesterase, acid-like 3B MonosaccharideMetabolism DIA1 22q13 NADH-cytochrome b5 reductase (diaphorase)Monosaccharide Metabolism GALK1 17q24 Galactokinase 1 MonosaccharideMetabolism LCT 2q21 Lactase Monosaccharide Metabolism KHK 2p23Ketohexokinase (fructokinase) Monosaccharide Metabolism PMM1 22q13Phosphomannomutase 1 Monosaccharide Metabolism FPGT 1p31Fucose-1-phosphate guanylyltransferase Monosaccharide Metabolism H6PD1p36 Hexose-6-phosphate dehydrogenese Pentose Phosphate PGD 1p36Phosphogluconate dehydrogenase Pentose Phosphate RPIA 2p11 Ribose5-phosphate Isomerase A Pentose Phosphate RBKS 2p23 Ribokinase PentosePhosphate TALDO1 11p15 Transaldolase 1 Pentose Phosphate GYS1 19q13Glycogen synthase 1 (muscle) Starch/Sucrose Metabolism AGL 1p21Amylo-1,6-glucosidase, 4-alpha-glucanotransferase Starch/SucroseMetabolism TESK2 1p32 Testis-specific kinase 2 Starch/Sucrose MetabolismAMY1A 1p21 Salivary alpha-amylase Starch/Sucrose Metabolism RUVBL2 19q13RuvB-like 2 Starch/Sucrose Metabolism GAL3ST1 22q12Galactose-3-O-sulfotransferase 1 Glycolipid POMT2 14q24Protein-O-mannosyltransferase 2 O-linked mannose POMGNT1 1p34.1 GlcNActransferase O-linked mannose SLC35A4 5q31.3 UDP-Galactose TransporterSugar Nucleotide Transport SLC35D1 1p31-p32 UDP-GalNAc Transporter SugarNucleotide Transport SLC35B1 17q21.33 UDP-Galactose Transporter SugarNucleotide Transport B) Genes that do not map to 18 MS Loci in Table 5B3GNT3 19p13.1 β1,3 N-acetylglucosominyltransferase III O-glycan, GPIB3GNT5 3q28 β1,3 N-acetylglucosominyltransferase V Glycolipid B4GALT19q13 β1,4 Galactosyltransferase I ? B4GALT4 3q13.3 β1,4Galactosyltransferase IV Glycolipid, N-Glycan B4GALT5 20q13.1-13.2 β1,4Galactosyltransferase V N-Glycan B4GALT6 18q11 β1,4Galactosyltransferase IV Glycolipd B3GALT3 3q25 β1,3Galactosyltransferase III N-,O-Glycan B3GALT4 6p21 β1,3Galactosyltransferase IV N-,O-Glycan B3GALT5 21q22.3 β1,3Galactosyltransferase V N-,O-Glycan GALNT1 18q12.1N-Acetylgalactosaminyltransferase I O-Glycan GALNT6 12q13N-Acetylgalactosaminyltransferase VI O-Glycan GALNT8 12p13.3N-Acetylgalactosaminyltransferase VIII O-Glycan GALNT11 7q34-q36N-Acetylgalactosaminyltransferase XI O-Glycan GALNT12 9q31.1N-Acetylgalactosaminyltransferase XII O-Glycan GALNT15 7q36.2N-Acetylgalactosaminyltransferase XV O-Glycan GALGT 12q13N-Acetylgalactosaminyltransferase Glycolipid ChGn 8p21.3N-Acetylgalactosaminyltransferase Glycosaminoglycan SIAT1 3q27-q28 α2,6Siayltransferase I (ST6Gal I) N-Glycan SIAT4A 8q24.22 α2,3Siayltransferase I (ST3Gal IA) O-Glycan SIAT4B 16q22 α2,3Siayltransferase II (ST3Gal II) Glycolipid, O-Glycan SIAT4C 11q23-q24α2,3 Siayltransferase IV (ST3Gal IV) O-Glycan SIAT7D & F 9q34 α2,6Siayltransferase O-Glycan, glycolipid SIAT8A 12p11.2-p12.1 α2,8Siayltransferase I (ST8Sia I) Glycolipid SIAT8B 15q26 α2,8Siayltransferase II (ST8Sia II) N-Glycan SIAT8C 18q21.31 α2,8Siayltransferase III (ST8Sia III) N-Glycan SIAT8E 15q21.1 α2,8Siayltransferase V (ST8Sia V) Glycolipid SIAT10 3q12.2 α2,3Siayltransferase (ST3 VI) N-,O-Glycan, Glycolipid FUT4 11q21 α1,3Fucosyltransferase IV (FucT IV) ? FUT7 9q34.3 α1,3 FucosyltransferaseVII (FucT VII) ? FUT9 6q16 α1,3 Fucosyltransferase IX (FucT IX) ? FUT108p12 α1,3 Fucosyltransferase ? FUT11 10q22.3 α1,3 Fucosyltransferase ?OGT Xq13 O-linked N-acetylglucosaminyltransferase O-GlcNAc ABO9q34.1-q34.2 ABO Blood Group Transferase A and B ABO Blood group GCNT19q13 Core2 N-acetylglucosaminyltransferase I O-glycan GCNT2 6p24 Core2N-acetylglucosaminyltransferase II O-Glycan GCNT3 15q21.3 Core2N-acetylglucosaminyltransferase III O-Glycan EDEM1 3p26.1 ER degradationenhancer, mannosidase-alpha-like 1 N-Glycan MANBA 4q22-25 mannosidase,beta A, lysosomal N-Glycan KIAA0935 4p16.2 mannosidase, alpha calss 2Bmember 2 N-Glycan MANEA 6q16.2 mannosidase, endo alpha (eM) N-GlycanMAN1B1 9q34 mannosidase, alpha, class 1B, member I (ER M1) N-GlycanMAN2C1 15q11-q13 mannosidase, alpha, class 2C, member 1 N-Glycan MAN2B119p13 mannosidase, alpha, class 2B, member 1 N-Glycan MANBAL 20q11-q12mannosidase, beta A, lysosomal like N-Glycan SMP3 3q29 GPI-linkedmannosyltransferase GPI DPM1 20q13 Dol-P-mannosyltransferase 1 GPI DPM29q34.13 Dol-P-mannosyltransferase 2 GPI LOC200810 3q21.2 Similar to β1-4mannosyltransferase ? & LOC285407 LOC339879 3p14.1 Similar to β1-4mannosyltransferase ? LOC285544 4p16.1 Similar to β1-4mannosyltransferase ? & LOC391613 LOC401305 7p22.2 Similar to β1-4mannosyltransferase ? LOC200810 3q21 Similar to β1-4 mannosyltransferase? LOC392191 8p23.1 Similar to β1-4 mannosyltransferase ? & LOC392199LOC401712 12p13 Similar to β1-4 mannosyltransferase ? UGCG 9q31 CerimideGlucosyltransferase Glycolipid HS3ST1 4p16 Heparin Sulfate3-O-sulfotransferase I GAG HS3ST3A1 & B1 17p12-p11 Heparin Sulfate3-O-sulfotransferase IIIA GAG HS3ST4 16p11.2 Heparin Sulfate3-O-sulfotransferase IV GAG HS3ST5 6p22.31 Heparin Sulfate3-O-sulfotransferase V GAG HS6ST2 Xq26.2 Heparin Sulfate6-O-sulfotransferase II GAG HS6ST3 13q32.1 Heparin Sulfate6-O-sulfotransferase III GAG CHST1 11p11.2-p11.1 Keratan SulfateGal-6-Sulfotransferase GAG CHST2 3q24N-Acetylglucosaminyltransferase-6-O-Sulfotransferase II ? CHST3 10q22.2Chondroitin-6-O-sulfotransferase III GAG CHST4, 5 & 6 16q22N-Acetylglucosaminyltransferase-6-O-Sulfotransferase IV GAG CHST7Xp11.23 N-Acetylglucosaminyltransferase-6-O-Sulfotransferase VII ? CHST918q11.2 N-Acetylgalactoasaminyltransferase-4-O-Sulfotransferase IX ?CHST10 2q12.1 Carbohydrate Sulfotransferase X ? CHST12 7p22Chondroitin-4-O-Sulfotransferase XII GAG NDST2 10q22N-deacetylase/N-sulfotransferase II GAG NDST3 4q27N-deacatylase/N-sulfotransferase III GAG NDST4 4q25-26N-deacetylase/N-sulfotransferase IV GAG UGCG 9q31 UDP-Glucose Ceramideglucosyltransferase GAG B3GAT1 11q25 β1,3 Glucuronyltransferase I GAGB3GAT2 6q13 β1,3 Glucuronyltransferase II GAG XYLT1 16p12Xylosetransferase I GAG EXT1 8q36Glucuronyl/N-acetylglucosaminyltransferase I GAG EXT2 11p11-p12Glucuronyl/N-acetylglucosaminyltransferase II GAG EXTL3 8p21 α1,4 GlcNActranferase GAG GLCE 15q22.31 Glucuronyl C5 Epimerase GAG IDS Xq28Iduronate-2-sulfatase GAG IDUA 4p16.3 Iduronadase GAG HEXA 15q23-q24Hexosamindase GAG GUSB 7q21.11 Glucuronidase GAG GNS 12q14N-acetylglucosamine-6-sulfatase GAG GALNS 16q24.3N-acetylgalactosamine-6-sulfatase GAG GLB1 3p21.33 β Galactosidase GAG,Glycolipid GLA Xq22 α Galactosidase Glycolipid GBA2 9p13 β GlucosidaseII GBA3 4p15.31 β Glucosidase GANC 15q15.2 α Glucosidase, neutral CFUCA2 6q24 Fucosidase (plasma) NEU1 6p21.3 Sialidase (lysosomal) AGA4q32-33 Aspartylglucosaminidase N-glycan degradation GNE 9p13.1 ManNAcKinase/UDP-GlcNAc 2 epimerase Sugar-nucleotide RENBP Xq28 Renin bindingprotein Sugar-nucleotide GMPPB 2q36.1 GDP-mannose pyrophosphorylaseSugar-nucleotide FBP1 9q22.3 Fructose 1,6-bisphosphatase Glycolysis FBP29q22.3 Fructose 1,6-bisphosphatase Glycolysis PFKL 21q22Phosphofructokinase-liver Glycolysis PFKM 12q13Phosphofructokinase-muscle Glycolysis PFKX 12ptel Phosphofructokinase-XGlycolysis ALDOA 16q22-q24 Aldolase A Glycolysis ALDOB 9q21-q22 AldolaseB Glycolysis ALDOC 17cent-q12 Aldolase C Glycolysis GAPD 12p13Glyceraldehyde-3-phosphate dehydrogenase Glycolysis PGK1 Xq13Phosphoglycerate Kinase 1 Glycolysis PGK2 6p12 Phosphoglycerate Kinase 2Glycolysis PGAM1 10q25 Phosphoglyceromutase 1 Glycolysis PGAM2 7p12-p13Phosphoglyceromutase 2 Glycolysis ENO2 12p13 Enolase 2 Glycolysis ENO317pter-p11 Enolase 3 Glycolysis PDHA1 Xp22 Pyruvate dehydrogenase(lipoamide) alpha 1 Glycolysis DLAT 11q23 DihydrolipoamideS-acetyltransferase Glycolysis DLD 7q31-q32 Dihydrolipoamidedehydrogenase Glycolysis ACAS2 20q11 Acetyl-Coenzyme A synthetase 2Glycolysis ALDH1A1 9q21 Aldehyde dehydrogenase 1 family, member A1Glycolysis ALDH3A1 17p11 Aldehyde dehydrogenase 3 family, member A1Glycolysis ADH1A 4q21-q23 Alcohol dehydrogenase 1A (class I), alphapolypeptide Glycolysis C) Genes that do not map to 18 MS Loci in Table 5MTMR1 Xq28 Myotubularin related protein 1 Monosaccharide Metabolism NANS9p23-p24 Sialic acid synthase Monosaccharide Metabolism CMAS 12p12Cytidine monophospho-N-acetylneuraminic acid synthetase MonosaccharideMetabolism GLB1 3p21 Beta-galactosidase 1 Monosaccharide Metabolism GLAXq22 Alpha-galactosidase Monosaccharide Metabolism AKR1B1 7q35 Aldo-ketoreductase family 1, member B1 Monosaccharide Metabolism UGT2B11 4q13 UDPglycosyltransferase 2 family, polypeptide B11 Monosaccharide MetabolismGMDS 6p25 GDP-mannose 4,6-dehydratase Monosaccharide Metabolism UGDH4p15 UDP-glucose dehydrogenase Monosaccharide Metabolism SORD 15q15Sorbitol dehydrogenase Monosaccharide Metabolism MPI 15q22 Mannosephosphate isomerase Monosaccharide Metabolism TSTA3 8q24 Tissue specifictransplantation antigen P35B Monosaccharide Metabolism FUK 16q22Fucokinase Monosaccharide Metabolism TPI1 12p13 Triosephosphateisomerase 1 Monosaccharide Metabolism PFKFB1 Xp116-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 MonosaccharideMetabolism G6PD Xq28 Glucose-6-phosphate dehydrogenase Pentose PhosphatePGLS 19p13 6-Phosphogluconolactonase Pentose Phosphate TKT 3p14Transketolase Pentose Phosphate PRPS1 Xq21-q27 Phosphoribosylpyrophosphate synthetase 1 Pentose Phosphate GALT 9p13Galactose-1-phosphate uridylyltransferase Sugar nucleotide TGDS 13q32TDP-glucose 4,6-dehydratase Sugar nucleotide LTB4DH 9q32 Leukotriene B412-hydroxydehydrogenase Sugar nucleotide SI 3q25-q26 Sucrase-isomaltaseStarch/Sucrose Metabolism ENPP1 6q22-q23 Ectonucleotidepyrophosphatase/phosphodiesterase 1 Starch/Sucrose Metabolism PYGB 20p11Glycogen phosphorylase (brain) Starch/Sucrose Metabolism GBE1 3p12Glucan (1,4-alpha-), branching enzyme 1 Starch/Sucrose MetabolismUGT2B11 4q13 UDP glycosyltransferase 2 family, polypeptide B11Starch/Sucrose Metabolism TREH 11q23 Trehalase Starch/Sucrose MetabolismMGAM 7q34 Maltase-glucoamylase Starch/Sucrose Metabolism SLC35A2 Xp11UDP-galactose transporter Sugar nucleotide transport SLC35A1 6q15CMP-Sialic Acid transporter Sugar nucleotide transport SLC35A5 3q13.2Solute carrier family 35, member A5 Sugar nucleotide transport SLC35B36p24.3 Solute carrier family 35, member B3 Sugar nucleotide transportSLC35B4 7q33 Solute carrier family 35, member B4 Sugar nucleotidetransport SLC35C1 11p11.2 GDP-fucose transporter 1 Sugar nucleotidetransport SLC3BE3 12q15 Solute carrier family 35, member E3 Sugarnucleotide transport

TABLE 7 Table Association of MGAT1 SNPV with Multiple Sclerosis andRheumatoid Arthritis Case Control Transmission Disequilbrium Test (TDT)Subjects Transmissions SNP+:SNP− p-value OR 95% CI Observed Expected TDTChi² p-value Multiple Sclerosis 21:103 0.0004 4.52 1.85-11.0 {closeoversize parenthesis} 12 8 4.0 0.046 Rheumatoid Arthritis 8:39 0.00684.54 1.51-10.3 Thyroid Autoimmunity 10:103 0.101 — — 8 9.5 0.474 0.491Control  7:155 For case control the Odds Ratio, P values and 95%Confidence Intervals were calculated with Contingency Tables andFisher's Exact Test (1 tailed, FET). For TDT, over 300 parent sets withat least one affected child were screened for MGAT1 SNP V. Affectedchildren of MGAT1 SNP V heterozygous parents were then genotyped andused to calculate the TDT Chi2 statistitic and p-value. Due too therarity of parents positive for MGAT1 SNP V and the similar strength ofassociation (ie OR) of Multiple Sclerosis and Rheumatoid Arthritis bycase control, affected children with these two diseases were groupedtogether for purposes of transmission in the TDT. All subjects areCaucasian. Control subjects for case control did not have personalhistory of autoimmune disease.

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What is claimed:
 1. A method for treating insulin-dependent diabetesmellitus comprising administering to a subject having insulin-dependentdiabetes mellitus a pharmaceutical composition comprisingN-acetylglucosamine (GlcNAc) and a pharmaceutically acceptable carrier,wherein the level of Mgat5 modified glycans is increased in the subject,thereby treating the subject's insulin-dependent diabetes mellitus. 2.The method of claim 1, further comprising administering a hexosaminepathway metabolite.
 3. The method of claim 2, wherein GlcNAc and thehexosamine pathway metabolite are administered in synergisticquantities.
 4. The method of claim 2, further comprising administeringuridine, uridine 5′ diphosphate (UDP), uridine 5′ monophosphate (UMP),uridine 5′ triphosphate (UTP), glutamine or uracil.
 5. The method ofclaim 4, further comprising administering Vitamin D3.
 6. The method ofclaim 2, wherein the composition is administered orally.
 7. The methodof claim 1, further comprising administering uridine, uridine 5′diphosphate (UDP), uridine 5′ monophosphate (UMP), uridine 5′triphosphate (UTP), glutamine or uracil.
 8. The method of claim 7,further comprising administering Vitamin D3.
 9. The method of claim 1,wherein the composition is administered orally.
 10. A method fortreating insulin-dependent diabetes mellitus comprising administering toa subject having insulin-dependent diabetes mellitus a pharmaceuticalcomposition comprising N-acetylglucosamine (GlcNAc) and a compoundselected from uridine, uridine 5′ diphosphate (UDP), uridine 5′monophosphate (UMP), uridine 5′ triphosphate (UTP), glutamine or uracil,and a pharmaceutically acceptable carrier, wherein the level of Mgat5modified glycans is increased in the subject, thereby treating thesubject's insulin-dependent diabetes mellitus.
 11. The method of claim10, further comprising administering Vitamin D3.
 12. The method of claim11, wherein the composition is administered orally.
 13. The method ofclaim 10, wherein the composition is administered orally.